U.S. patent number 7,189,397 [Application Number 10/647,818] was granted by the patent office on 2007-03-13 for cytotoxicity mediation of cells evidencing surface expression of cd44.
This patent grant is currently assigned to Arius Research Inc.. Invention is credited to Helen P. Findlay, Susan E. Hahn, David S. F. Young.
United States Patent |
7,189,397 |
Young , et al. |
March 13, 2007 |
Cytotoxicity mediation of cells evidencing surface expression of
CD44
Abstract
This invention relates to the diagnosis and treatment of
cancerous diseases, particularly to the mediation of cytotoxicity
of tumor cells; and most particularly to the use of cancerous
disease modifying antibodies (CDMAB), optionally in combination
with one or more chemotherapeutic agents, as a means for initiating
the cytotoxic response. The invention further relates to binding
assays which utilize the CDMABs of the instant invention.
Inventors: |
Young; David S. F. (Toronto,
CA), Hahn; Susan E. (Toronto, CA), Findlay;
Helen P. (Toronto, CA) |
Assignee: |
Arius Research Inc. (Ontario,
CA)
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Family
ID: |
34216605 |
Appl.
No.: |
10/647,818 |
Filed: |
August 22, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050008646 A1 |
Jan 13, 2005 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10603000 |
Jun 23, 2003 |
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09727361 |
Dec 2, 2003 |
6657048 |
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09415278 |
Oct 8, 1999 |
6180357 |
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Current U.S.
Class: |
424/156.1;
424/181.1; 435/7.23; 530/387.3; 530/388.1; 530/388.22; 530/388.85;
530/391.3; 530/391.7; 435/70.21; 435/69.6; 424/155.1; 424/143.1;
424/141.1; 424/133.1 |
Current CPC
Class: |
G01N
33/57423 (20130101); G01N 33/57419 (20130101); G01N
33/57449 (20130101); G01N 33/57484 (20130101); A61P
35/00 (20180101); G01N 33/57415 (20130101); C07K
16/2884 (20130101); A61K 49/0058 (20130101); G01N
33/56972 (20130101); C07K 16/00 (20130101); C07K
16/3015 (20130101); A61K 49/0041 (20130101); C07K
16/30 (20130101); A61K 2039/505 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); G01N 33/574 (20060101); C07K
16/00 (20060101); C12P 21/04 (20060101); C12P
21/08 (20060101) |
Field of
Search: |
;424/130.1,133.1,141.1,143.1,155.1,181.1,183.1,156.1,7.23,69.6,70.21
;530/387.1,387.3,388.1,388.22,388.8,397.7,388.85,391.3,391.7 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 9412631 |
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Jun 1994 |
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WO |
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WO02/094879 |
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Nov 2002 |
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WO |
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Primary Examiner: Huff; Sheela J.
Assistant Examiner: Blanchard; David J.
Attorney, Agent or Firm: McHale & Slavin, P.A.
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of application Ser. No.
10/603,000, filed June 23, 2003, which is a continuation-in-part of
application Ser. No. 09/727,361, filed Nov. 29, 2000, now U.S. Pat.
No. 6,657,048, issued Dec. 2, 2003, which is a continuation-in-part
of application Ser. No. 09/415,278, filed Oct. 8, 1999, now U.S.
Pat. No. 6,180,357 B1, the contents of each of which are herein
incorporated by reference.
Claims
What is claimed is:
1. A process for mediating cytotoxicity of a human tumor cell which
expresses CD44 on the cell surface, which is recognized by the
isolated monoclonal antibody produced by the hybridoma deposited
with the ATCC as PTA-4621 or an antigen binding fragment produced
from said isolated monoclonal antibody comprising: contacting said
human tumor cell with said isolated monoclonal antibody or said
antigen binding fragment, whereby cytotoxicity occurs as a result
of binding of said isolated monoclonal antibody or said antigen
binding fragment with said CD44.
2. The process of claim 1 wherein the human tumor cell is contacted
with a humanized antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the ATCC under Accession
Number PTA-4621 or an antigen binding fragment produced from said
humanized antibody.
3. The process of claim 1 wherein said isolated monoclonal antibody
or said antigen binding fragment are conjugated with a member
selected from the group consisting of cytotoxic moieties, enzymes,
radioactive compounds, and hematogenous cells.
4. The process of claim 1 wherein the human tumor cell is contacted
with a chimeric antibody of the isolated monoclonal antibody
produced by the hybridoma deposited with the ATCC under Accession
Number PTA-4621 or an antigen binding fragment produced from said
chimeric antibody.
5. The process of claim 1 wherein the human tumor cell is selected
from the group consisting of colon, ovarian, lung, and breast.
6. A binding assay to determine the presence of cells which express
CD44 which is specifically recognized by the isolated monoclonal
antibody produced by the hybridoma deposited with the ATCC as
PTA4621, or an antigen binding fragment produced from said isolated
monoclonal antibody comprising: providing a cell sample; providing
the isolated monoclonal antibody produced by the hybridoma
deposited with the ATCC as PTA-4621 or said antigen binding
fragment produced from the isolated monoclonal antibody; contacting
said isolated monoclonal antibody or said antigen binding fragment
with said cell sample; and determining binding of said isolated
monoclonal antibody or antigen binding fragment thereof with said
cell sample; whereby the presence of cells which express CD44 which
is specifically recognized by said isolated monoclonal antibody or
said antigen binding fragment is determined.
7. The binding assay of claim 6 wherein the cell sample is selected
from the group consisting of colon, ovarian, lung, and breast
tissue.
8. A method of extending survival and delaying tumor growth in a
mammal, wherein said tumor expresses CD44 which is specifically
recognized by the isolated monoclonal antibody produced by the
hybridoma deposited with the ATCC as PTA-4621 comprising
administering to said mammal said isolated monoclonal antibody or
an antigen binding fragment produced from said isolated monoclonal
antibody in an amount effective to reduce said mammal's tumor
burden, whereby tumor growth is delayed and survival is
extended.
9. The method of claim 8 wherein said isolated monoclonal antibody
or said antigen binding fragment is conjugated to a cytotoxic
moiety.
10. The method of claim 9 wherein said cytotoxic moiety is a
radioactive isotope.
11. The method of claim 8 wherein said isolated monoclonal antibody
activates complement.
12. The method of claim 8 wherein said isolated monoclonal antibody
mediates antibody dependent cellular cytotoxicity.
13. The method of claim 8 wherein the antibody administered is a
humanized antibody of the isolated monoclonal antibody produced by
the hybridoma deposited with the ATCC under Accession Number
PTA4621 or an antigen binding fragment produced from said humanized
antibody.
14. The method of claim 8 wherein the antibody administered is a
chimeric antibody of the isolated monoclonal antibody produced by
the hybridoma deposited with the ATCC under Accession Number
PTA-4621 or an antigen binding fragment produced from said chimeric
antibody.
15. The binding assay of claim 6 wherein said cell sample is
contacted with a humanized antibody of the isolated monoclonal
antibody produced by the hybridoma deposited with the ATCC under
Accession Number PTA-4621 or an antigen binding fragment produced
from said humanized antibody.
16. The binding assay of claim 6 wherein said cell sample is
contacted with a chimeric antibody of the isolated monoclonal
antibody produced by the hybridoma deposited with the ATCC under
Accession Number PTA-4621 or an antigen binding fragment produced
from said chimeric antibody.
Description
FIELD OF THE INVENTION
This invention relates to the diagnosis and treatment of cancerous
diseases, particularly to the mediation of cytotoxicity of tumor
cells; and most particularly to the use of cancerous disease
modifying antibodies (CDMAB), optionally in combination with one or
more chemotherapeutic agents, as a means for initiating the
cytotoxic response. The invention further relates to binding assays
which utilize the CDMAB of the instant invention.
BACKGROUND OF THE INVENTION
Raising monoclonal antibodies against human white blood cells led
to the discovery of the CD44 antigen; a single chain hyaluronic
acid (HA) binding glycoprotein expressed on a wide variety of
normal tissue and on all types of hematopoietic cells. It was
originally associated with lymphocyte activation and homing.
Currently, its putative physiological role also includes activation
of inflammatory genes, modulation of cell cycle, induction of cell
proliferation, induction of differentiation and development,
induction of cytoskeletal reorganization and cell migration and
cell survival/resistance to apoptosis.
In humans, the single gene copy of CD44 is located on the short arm
of chromosome 11, 11p13. The gene contains 19 exons; the first 5
are constant, the next 9 are variant, the following 3 are constant
and the final 2 are variant. Differential splicing can lead to over
1000 different isoforms. However, currently only several dozen
naturally occurring variants have been identified.
The CD44 standard glycoprotein consists of a N-terminal
extracellular (including a 20 a.a. leader sequence, and a membrane
proximal region (85 a.a.)) domain (270 a.a.), a transmembrane
region (21 a.a.) and a cytoplasmic tail (72 a.a.). The
extracellular region also contains a link module at the N-terminus.
This region is 92 a.a. in length and shows homology to other HA
binding link proteins. There is high homology between the mouse and
human forms of CD44. The variant forms of the protein are inserted
to the carboxy terminus of exon 5 and are located extracellularly
when expressed.
A serum soluble form of CD44 also occurs naturally and can arise
from either a stop codon (within the variable region) or from
proteolytic activity. Activation of cells from a variety of stimuli
including TNF-.alpha. results in shedding of the CD44 receptor.
Shedding of the receptor has also been seen with tumor cells and
can result in an increase in the human serum concentration of CD44
by up to 10-fold. High CD44 serum concentration suggests malignancy
(ovarian cancer being the exception).
The standard form of CD44 exists with a molecular weight of
approximately 37 kD. Post-translational modifications increase the
molecular weight to 80 90 kD. These modifications include amino
terminus extracellular domain N-linked glycosylations at asparagine
residues, O-linked glycosylations at serine/threonine residues at
the carboxy terminus of the extracellular domain and
glycosaminoglycan additions. Splice variants can range in size from
80 250 kD.
HA, a polysaccharide located on the extracellular matrix (ECM) in
mammals, is thought to be the primary CD44 ligand. However, CD44
has also been found to bind such proteins as collagen, fibronectin,
laminin etc. There appears to be a correlation between HA binding
and glycosylation. Inactive CD44 (does not bind HA) has the highest
levels of glycosylation, active CD44 (binding HA) the lowest while
inducible CD44 (does not or weakly binds HA unless activated by
cytokines, monoclonal antibodies, growth factors, etc.) has
glycoslyation levels somewhere in between the active and inactive
forms.
CD44 can mediate some of its functions through signal transduction
pathways that depend on the interaction of the cell, stimulus and
the environment. Some of these pathways include the NF.kappa.B
signaling cascade (involved in the inflammatory response), the
Ras-MAPK signal transduction pathway (involved with activating cell
cycling and proliferation), the Rho family of proteins (involved
with cytoskeleton reorganization and cell migration) and the
PI3-K-related signaling pathway (related to cell survival). All of
the above-mentioned functions are closely associated with tumor
disease initiation and progression. CD44 has also been implicated
in playing a role in cancer through a variety of additional
mechanisms. These include the presentation of growth factors,
chemokines and cytokines by cell surface proteoglycans present on
the cell surface of CD44 to receptors involved in malignancy. Also,
the intracellular degradation of HA by lysosomal hylauronidases
after internalization of the CD44-HA complex can potentially
increase the likelihood of tumor invasiveness and induction of
angiogenesis through the ECM. In addition, the transmission of
survival or apoptotic signals has been shown to occur through
either the standard or variable CD44 receptor. CD44 has also been
suggested to be involved in cell differentiation and migration.
Many, if not all, of these mechanisms are environment and cell
dependent and several give rise to variable findings. Therefore,
more research is required before any conclusions can be drawn.
In order to validate a potential functional role of CD44 in cancer,
expression studies of CD44 were undertaken to determine if
differential expression of the receptor correlates with disease
progression. However, inconsistent findings were observed in a
majority of tumor types and this is probably due to a combination
of reagents, technique, pathological scoring and cell type
differences between researchers. Renal cell carcinoma and
non-Hodgkin's lymphoma appear to be the exception in that patients
with high CD44 expressing tumors consistently had shorter survival
times than their low or non-CD44 expressing counterparts.
Due to its association with cancer, CD44 has been the target of the
development of anti-cancer therapeutics. There is still controversy
as to whether the standard or the variant forms of CD44 are
required for tumor progression. There is in vivo animal data to
support both views and again it may be tumor type and even cell
type dependent. Different therapeutic approaches have included
injection of soluble CD44 proteins, hyaluronan synthase cDNA,
hyaluronidase, the use of CD44 antisense and CD44 specific
antibodies. Each approach has led to some degree of success thereby
providing support for anti-CD44 cancer therapeutics.
Both variant and standard CD44 specific monoclonal antibodies have
been generated experimentally but for the most part these
antibodies have no intrinsic biological activity, rather they bind
specifically to the type of CD44 they recognize However, there are
some that are either active in vitro or in vivo but generally not
both. Several anti-CD44 antibodies have been shown to mediate
cellular events. For example the murine antibody A3D8, directed
against human erythrocyte Lutheran antigen CD44 standard form, was
shown to enhance CD2 (9-1 antibody) and CD3 (OKT3 antibody)
mediated T cell activation; another anti-CD44 antibody had similar
effects. A3D8 also induced IL-1 release from monocytes and IL-2
release from T lymphocytes. Interestingly, the use of A3D8 in
conjunction with drugs such as daunorubicin, mitoxantrone and
etoposide inhibited apoptosis induction in HL60 and NB4 AML cells
by abrogating the generation of the second messenger ceramide. The
J173 antibody, which does not have intrinsic activity and is
directed against a similar epitope of CD44s, did not inhibit
drug-induced apoptosis. The NIH44-1 antibody, directed against an
85 110 KD and 200 KD form of CD44, augmented T-cell proliferation
through a pathway the authors speculated as either cross-linking or
aggregation of CD44. Taken together, there is no evidence that
antibodies such as these are suitable for use as cancer
therapeutics since they either are not directed against cancer
(e.g. activate lymphocytes), induce cell proliferation, or when
used with cytotoxic agents inhibited drug-induced death of cancer
cells.
Several anti-CD44 antibodies have been described which demonstrate
anti-tumor effects in vivo. The antibody 1.1ASML, a mouse anti-rat
IgG1 directed to the v6 variant of CD44, has been shown to decrease
the lymph node and lung metastases of the rat pancreatic
adenocarcinoma BSp73ASML. Survival of the treated animals was
concomitantly increased. The antibody was only effective if
administered before lymph node colonization, and was postulated to
interfere with cell proliferation in the lymph node. There was no
direct cytototoxicy of the antibody on the tumor cells in vitro,
and the antibody did not enhance complement-mediated cytotoxicity,
or immune effector cell function. Utility of the antibody against
human cells was not described.
Breyer et al. described the use of a commercially-available
antibody to CD44s to disrupt the progression of an
orthotopically-implanted rat glioblastoma. The rat glioblastoma
cell line C6 was implanted in the frontal lobe, and after 1 week,
the rats were given 3 treatments with antibody by intracerebral
injection. Treated rats demonstrated decreased tumor growth, and
higher body weight than buffer or isotype-control treated rats. The
antibody was able to inhibit adhesion of cells in vitro to
coverslips coated with extracellular matrix components, but did not
have any direct cytotoxic effects on cells. This antibody was not
tested against human cells.
A study was carried out which compared the efficacy of an antibody
to CD44s (IM-7.8.1) to an antibody to CD44v10 (K926). The highly
metastatic murine melanoma line B16F10, which expresses both CD44
isoforms, was implanted i.v. into mice. After 2 days, antibodies
were given every third day for the duration of the study. Both
antibodies caused a significant reduction of greater than 50% in
the number of lung metastases; there was no significant difference
in efficacy between the two antibodies. The antibody did not affect
proliferation in vitro, and the authors, Zawadzki et al, speculated
that the inhibition of tumor growth was due to the antibody
blocking the interaction of CD44 with its ligand. In another study
using IM-7.8.1, Zahalka et al demonstrated that the antibody and
its F(ab').sub.2 fragment were able to block the lymph node
infiltration by the murine T-cell lymphoma LB. This conferred a
significant survival benefit to the mice. Wallach-Dayan et al
showed that transfection of LB-TRs murine lymphoma, which does not
spontaneously form tumors, with CD44v4-v10 conferred the ability to
form tumors. IM-7.8.1 administration decreased tumor size of the
implanted transfected cells in comparison to the isotype control
antibody. None of these studies demonstrated human utility for this
antibody.
GKW.A3, a mouse IgG2a, is specific for human CD44 and prevents the
formation and metastases of a human melanoma xenograft in SCID
mice. The antibody was mixed with the metastastic human cell line
SMMU-2, and then injected subcutaneously. Treatments were continued
for the following 3 weeks. After 4 weeks, only 1 of 10 mice
developed a tumor at the injection site, compared to 100% of
untreated animals. F(ab').sub.2 fragments of the antibody
demonstrated the same inhibition of tumor formation, suggesting
that the mechanism of action was not dependent on complement or
antibody-dependent cellular cytotoxicity. If the tumor cells were
injected one week prior to the first antibody injection, 80% of the
animals developed tumors at the primary site. However, it was noted
that the survival time was still significantly increased. Although
the delayed antibody administration had no effect on the primary
tumor formation, it completely prevented the metastases to the
lung, kidney, adrenal gland, liver and peritoneum that were present
in the untreated animals. This antibody does not have any direct
cytotoxicity on the cell line in vitro or interfere with
proliferation of SMMU-2 cells, and appears to have its major effect
on tumor formation by affecting metastasis or growth. One notable
feature of this antibody was that it recognized all isoforms of CD
44, which suggests limited possibilities for therapeutic use.
Strobel et al describe the use of an anti-CD44 antibody (clone 515)
to inhibit the peritoneal implantation of human ovarian cancer
cells in a mouse xenograft model. The human ovarian cell line 36M2
was implanted i.p. into mice in the presence of the anti-CD44
antibody or control antibody, and then treatments were administered
over the next 20 days. After 5 weeks, there were significantly
fewer nodules in the peritoneal cavity in the antibody treated
group. The nodules from both the anti-CD44 and control treated
groups were the same size, suggesting that once the cells had
implanted, the antibody had no effect on tumor growth. When cells
were implanted subcutaneously, there was also no effect on tumor
growth, indicating that the antibody itself did not have an
anti-proliferative or cytotoxic effect. In addition, there was no
effect of the antibody on cell growth in vitro.
VFF-18, also designated as BIWA 1, is a high-affinity antibody to
the v6 variant of CD44 specific for the 360 370 region of the
polypeptide. This antibody has been used as a
.sup.99mTechnetium-labelled conjugate in a Phase 1 clinical trial
in 12 patients. The antibody was tested for safety and targeting
potential in patients with squamous cell carcinoma of the head and
neck. Forty hours after injection, 14% of the injected dose was
taken up by the tumor, with minimal accumulation in other organs
including the kidney, spleen and bone marrow. The highly selective
tumor binding suggests a role for this antibody in
radioimmunotherapy, although the exceptionally high affinity of
this antibody prevented penetration into the deeper layers of the
tumor. Further limiting the application of BIWA 1 is the
immunogenicity of the murine antibody (11 of 12 patients developed
human anti-mouse antibodies (HAMA)), heterogenous accumulation
throughout the tumor and formation of antibody-soluble CD44
complexes. WO 02/094879 discloses a humanized version of VFF-18
designed to overcome the HAMA response, designated BIWA 4. BIWA 4
was found to have a significantly lower antigen binding affinity
than the parent VFF 18 antibody. Surprisingly, the lower affinity
BIWA 4 antibody had superior tumor uptake characteristics than the
higher affinity BIWA 8 humanized VFF-18 antibody. Both
.sup.99mTechnetium-labelled and .sup.186Rhenium-labelled BIWA 4
antibody were assessed in a 33 patient Phase 1 clinical trial to
determine safety, tolerability, tumor accumulation and maximum
tolerated dose, in the case of .sup.186Re-labelled BIWA 4. There
appeared to be tumor related uptake of .sup.99mTc-labelled BIWA 4.
There were no tumor responses seen with all doses of
.sup.186Re-labelled BIWA 4, although a number had stable disease;
the dose limiting toxicity occurred at 60 mCi/m.sup.2. There was a
50 65% rate of adverse events with 12 of 33 patients deemed to have
serious adverse events (thrombocytopenia, leucopenia and fever) and
of those 6, all treated with .sup.186Re-labelled BIWA 4, died in
the course of treatment or follow-up due to disease progression.
Two patients developed human anti-human antibodies (HAHA). A Phase
1 dose escalation trial of .sup.186Re-labelled BIWA 4 was carried
out in 20 patients. Oral mucositis and dose-limiting
thrombocytopenia and leucocytopenia were observed; one patient
developed a HAHA response. Stable disease was seen in 5 patients
treated at the highest dose of 60 mCi/m.sup.2. Although deemed to
be acceptable in both safety and tolerablility for the efficacy
achieved these studies have higher rates of adverse events compared
to other non-radioisotope conjugated biological therapies in
clinical studies. U.S. Patent Application US 2003/0103985 discloses
a humanized version of VFF-18 conjugated to a maytansinoid,
designated BIWI 1, for use in tumor therapy. A humanized VFF 18
antibody, BIWA 4, when conjugated to a toxin, i.e. BIWI 1, was
found to have significant anti-tumor effects in mouse models of
human epidermoid carcinoma of the vulva, squamous cell carcinoma of
the pharynx or breast carcinoma. The unconjugated version, BIWA 4,
did not have anti-tumor effects and the conjugated version, BIWI 1,
has no evidence of safety or efficacy in humans.
Mab U36 is a murine IgG1 antibody generated by UM-SCC-22B human
hypopharyngeal carcinoma cell immunization and selection for cancer
and tissue specificity. Antigen characterization through cDNA
cloning and sequence analysis identified the v6 domain of
keratinocyte-specific CD44 splice variant epican as the target of
Mab U36. Immunohistochemistry studies show the epitope to be
restricted to the cell membrane. Furthermore, Mab U36 labeled 94%
of the head and neck squamous cell carcinomas (HNSCC) strongly, and
within these tumors there was uniformity in cell staining. A 10
patient .sup.99mTc-labelled Mab U36 study showed selective
accumulation of the antibody to HNSCC cancers (20.4+/-12.4%
injected dose/kg at 2 days); no adverse effects were reported but
two patients developed HAMA. In a study of radio-iodinated murine
Mab U36 there were 3 cases of HAMA in 18 patients and selective
homogenous uptake in HNSCC. In order to decrease the antigenicity
of Mab U36 and decrease the rate of HAMA a chimeric antibody was
constructed. Neither the chimeric nor the original murine Mab U36
has ADCC activity. There is no evidence of native functional
activity of Mab U36. .sup.186Re-labelled chimeric Mab U36 was used
to determine the utility of Mab U36 as a therapeutic agent. In this
Phase 1 escalating dose trial 13 patients received a scouting dose
of .sup.99mTc-labelled chimeric Mab U36 followed by
.sup.186Re-labelled chimeric Mab U36. There were no acute adverse
events reported but following treatment dose limiting myelotoxcity
(1.5 GBq/m.sup.2) in 2 of 3 patients, and thrombocytopenia in one
patient treated with the maximum tolerated dose (1.0 GBq/m.sup.2)
were observed. Although there were some effects on tumor size these
effects did not fulfill the criteria for objective responses to
treatment. A further study of .sup.186Re-labelled chimeric Mab U36
employed a strategy of using granulocyte colony-stimulating factor
stimulated whole blood reinfusion to double the maximum-tolerated
activity to 2.8 Gy. In this study of nine patients with various
tumors of the head and neck 3 required transfusions for drug
related anemia. Other toxicity includes grade 3 myelotoxicity, and
grade 2 mucositis. No objective tumor responses were reported
although stable disease was achieved for 3 5 months in five
patients. Thus, it can be seen that although Mab U36 is a highly
specific antibody the disadvantage of requiring a
radioimmunoconjugate to achieve anti-cancer effects limits its
usefulness because of the toxicity associated with the therapy in
relation to the clinical effects achieved.
To summarize, a CD44v6 (1.1ASML) and CD44v10 (K926) monoclonal
antibody have been shown to reduce metastatic activity in rats
injected with a metastatic pancreatic adenocarcinoma or mice
injected with a malignant melanoma respectively. Another
anti-CD44v6 antibody (VFF-18 and its derivatives), only when
conjugated to a maytansinoid or a radioisotope, has been shown to
have anti-tumor effects. Anti-standard CD44 monoclonal antibodies
have also been shown to suppress intracerebral progression by rat
glioblastoma (anti-CD44s), lymph node invasion by mouse T cell
lymphoma (IM-7.8.1) as well as inhibit implantation of a human
ovarian cancer cell line in nude mice (clone 515), lung metastasis
of a mouse melanoma cell line (IM-7.8.1) and metastasis of a human
melanoma cell line in SCID mice (GKW.A3). The radioisotope
conjugated Mab U36 anti-CD44v6 antibody and its derivatives had
anti-tumor activity in clinical trials that were accompanied by
significant toxicity. These results, though they are encouraging
and support the development of anti-CD44 monoclonal antibodies as
potential cancer therapeutics, demonstrate limited effectiveness,
safety, or applicability to human cancers.
Thus, if an antibody composition were isolated which mediated
cancerous cell cytotoxicity, as a function of its attraction to
cell surface expression of CD44 on said cells, a valuable
diagnostic and therapeutic procedure would be realized.
Prior Patents:
U.S. Pat. No. 5,750,102 discloses a process wherein cells from a
patient's tumor are transfected with MHC genes which may be cloned
from cells or tissue from the patient. These transfected cells are
then used to vaccinate the patient.
U.S. Pat. No. 4,861,581 discloses a process comprising the steps of
obtaining monoclonal antibodies that are specific to an internal
cellular component of neoplastic and normal cells of the mammal but
not to external components, labeling the monoclonal antibody,
contacting the labeled antibody with tissue of a mammal that has
received therapy to kill neoplastic cells, and determining the
effectiveness of therapy by measuring the binding of the labeled
antibody to the internal cellular component of the degenerating
neoplastic cells. In preparing antibodies directed to human
intracellular antigens, the patentee recognizes that malignant
cells represent a convenient source of such antigens.
U.S. Pat. No. 5,171,665 provides a novel antibody and method for
its production. Specifically, the patent teaches formation of a
monoclonal antibody which has the property of binding strongly to a
protein antigen associated with human tumors, e.g. those of the
colon and lung, while binding to normal cells to a much lesser
degree.
U.S. Pat. No. 5,484,596 provides a method of cancer therapy
comprising surgically removing tumor tissue from a human cancer
patient, treating the tumor tissue to obtain tumor cells,
irradiating the tumor cells to be viable but non-tumorigenic, and
using these cells to prepare a vaccine for the patient capable of
inhibiting recurrence of the primary tumor while simultaneously
inhibiting metastases. The patent teaches the development of
monoclonal antibodies which are reactive with surface antigens of
tumor cells. As set forth at col. 4, lines 45 et seq., the
patentees utilize autochthonous tumor cells in the development of
monoclonal antibodies expressing active specific immunotherapy in
human neoplasia.
U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen
characteristic of human carcinomas and not dependent upon the
epithelial tissue of origin.
U.S. Pat. No. 5,783,186 is drawn to anti-Her2 antibodies which
induce apoptosis in Her2 expressing cells, hybridoma cell lines
producing the antibodies, methods of treating cancer using the
antibodies and pharmaceutical compositions including said
antibodies.
U.S. Pat. No. 5,849,876 describes new hybridoma cell lines for the
production of monoclonal antibodies to mucin antigens purified from
tumor and non-tumor tissue sources.
U.S. Pat. No. 5,869,268 is drawn to a method for generating a human
lymphocyte producing an antibody specific to a desired antigen, a
method for producing a monoclonal antibody, as well as monoclonal
antibodies produced by the method. The patent is particularly drawn
to the production of an anti-HD human monoclonal antibody useful
for the diagnosis and treatment of cancers.
U.S. Pat. No. 5,869,045 relates to antibodies, antibody fragments,
antibody conjugates and single chain immunotoxins reactive with
human carcinoma cells. The mechanism by which these antibodies
function is 2-fold, in that the molecules are reactive with cell
membrane antigens present on the surface of human carcinomas, and
further in that the antibodies have the ability to internalize
within the carcinoma cells, subsequent to binding, making them
especially useful for forming antibody-drug and antibody-toxin
conjugates. In their unmodified form the antibodies also manifest
cytotoxic properties at specific concentrations.
U.S. Pat. No. 5,780,033 discloses the use of autoantibodies for
tumor therapy and prophylaxis. However, this antibody is an
anti-nuclear autoantibody from an aged mammal. In this case, the
autoantibody is said to be one type of natural antibody found in
the immune system. Because the autoantibody comes from "an aged
mammal", there is no requirement that the autoantibody actually
comes from the patient being treated. In addition the patent
discloses natural and monoclonal antinuclear autoantibody from an
aged mammal, and a hybridoma cell line producing a monoclonal
antinuclear autoantibody.
U.S. Pat. No. 5,916,561 discloses a specific antibody, VFF-18, and
its variants directed against the variant exon v6 of the CD44 gene.
This antibody is an improvement over the comparator antibody in
that it recognizes a human CD44 v6 variant rather than a rat CD44
v6 variant. In addition this antibody discloses diagnostic assays
for CD44 v6 expression. There was no in vitro or in vivo function
disclosed for this antibody.
U.S. Pat. No. 5,616,468 discloses a monoclonal antibody, Var3.1,
raised against a synthetic peptide containing a sequence encoded by
the human exon 6A of the CD44 gene. Specifically this antibody does
not bind to the 90 kD form of human CD44 and is distinguished from
the Hermes-3 antibody. A method for detection of the v6 variant of
CD44 is provided, as well as a method for screening and assaying
for malignant transformation based on this antigen. A method for
screening for inflammatory disease based on detecting the antigen
in serum is also provided.
U.S. Pat. No. 5,879,898 discloses a specific antibody that binds to
a 129 bp exon of a human CD44 variant 6 that produces a 43 amino
acid peptide. The monoclonal antibody is produced by a number of
hybridoma cell lines: MAK<CD44>M-1.1.12,
MAK<CD44>M-2.42.3, MAK<CD44>M-4.3.16. The antibody is
generated from a fusion protein that contains at least a
hexapeptide of the novel CD44 v6 amino acid sequence. Further,
there is a disclosure of an immunoassay for the detection of exon 6
variant that can be used as a cancer diagnostic. Significantly,
there is no in vitro or in vivo function of this antibody
disclosed.
U.S. Pat. No. 5,942,417 discloses a polynucleotide that encodes a
CD44 like polypeptide, and the method of making a recombinant
protein using the polynucleotide and its variants. Antibodies are
claimed to these polypeptides however there are no specific
examples and there are no deposited clones secreting such
antibodies. Northern blots demonstrate the appearance of the
polynucleotide in several types of tissues, but there is no
accompanying evidence that there is translation and expression of
this polynucleotide. Therefore, there is no evidence that there
were antibodies to be made to the gene product of this
polynucleotide, that these antibodies would have either in vitro or
in vivo function, and whether they would be relevant to human
cancerous disease.
U.S. Pat. No. 5,885,575 discloses an antibody that reacts with a
variant epitope of CD44 and methods of identifying the variant
through the use of the antibody. The isolated polynucleotide
encoding this variant was isolated from rat cells, and the
antibody, mAb1.1ASML, directed against this variant recognizes
proteins of molecular weight 120 kD, 150 kD, 180 kD, and 200 kD.
The administration of monoclonal antibody 1.1ASML delayed the
growth and metastases of rat BSp73ASML in isogenic rats.
Significantly 1.1ASML does not recognize human tumors as
demonstrated by its lack of reactivity to LCLC97 human large-cell
lung carcinoma. A human homolog was isolated from LCLC97 but no
equivalent antibody recognizing this homolog was produced. Thus,
although an antibody specific to a variant of rat CD44 was produced
and shown to affect the growth and metastasis of rat tumors there
is no evidence for the effect the this antibody against human
tumors. More specifically the inventors point out that this
antibody does not recognize human cancers.
SUMMARY OF THE INVENTION
The instant inventors have previously been awarded U.S. Pat. No.
6,180,357, entitled "Individualized Patient Specific Anti-Cancer
Antibodies" directed to a process for selecting individually
customized anti-cancer antibodies which are useful in treating a
cancerous disease. For the purpose of this document, the terms
"antibody" and "monoclonal antibody" (mAb) may be used
interchangeably and refer to intact immunoglobulins produced by
hybridomas (e.g. murine or human), immunoconjugates and, as
appropriate, immunoglobulin fragments and recombinant proteins
derived from immunoglobulins, such as chimeric and humanized
immunoglobulins, F(ab') and F(ab').sub.2 fragments, single-chain
antibodies, recombinant immunoglobulin variable regions (Fv)s,
fusion proteins etc. It is well recognized in the art that some
amino acid sequence can be varied in a plupertide without
significant effect on the structure or function of the protein. In
the molecular rearrangement of antibodies, modifications in the
nucleic or amino acid sequence of the backbone region can generally
be tolerated. These include, but are not limited to, substitutions
(preferred are conservative substitutions), deletions or additions.
Furthermore, it is within the purview of this invention to
conjugate standard chemotherapeutic modalities, e.g. radionuclides,
with the CDMAB of the instant invention, thereby focusing the use
of said chemotherapeutics. The CDMAB can also be conjugated to
toxins, cytotoxic moieties, enzymes e.g. biotin conjugated enzymes,
or hematogenous cells.
This application utilizes substantially the method for producing
patient specific anti-cancer antibodies as taught in the '357
patent for isolating hybridoma cell lines which encode for
cancerous disease modifying monoclonal antibodies. These antibodies
can be made specifically for one tumor and thus make possible the
customization of cancer therapy. Within the context of this
application, anti-cancer antibodies having either cell-killing
(cytotoxic) or cell-growth inhibiting (cytostatic) properties will
hereafter be referred to as cytotoxic. These antibodies can be used
in aid of staging and diagnosis of a cancer, and can be used to
treat tumor metastases as well as primary tumors.
The prospect of individualized anti-cancer treatment will bring
about a change in the way a patient is managed. A likely clinical
scenario is that a tumor sample is obtained at the time of
presentation, and banked. From this sample, the tumor can be typed
from a panel of pre-existing cancerous disease modifying
antibodies. The patient will be conventionally staged but the
available antibodies can be of use in further staging the patient.
The patient can be treated immediately with the existing antibodies
and/or a panel of antibodies specific to the tumor can be produced
either using the methods outlined herein or through the use of
phage display libraries in conjunction with the screening methods
herein disclosed. All the antibodies generated will be added to the
library of anti-cancer antibodies since there is a possibility that
other tumors can bear some of the same epitopes as the one that is
being treated. The antibodies produced according to this method may
be useful to treat cancerous disease in any number of patients who
have cancers that bind to these antibodies.
Using substantially the process of U.S. Pat. No. 6,180,357, the
mouse monoclonal antibody H460-16-2 was obtained following
immunization of mice with cells from a patient's lung tumor biopsy.
The H460-16-2 antigen was expressed on the cell surface of a broad
range of human cell lines from different tissue origins. The breast
cancer cell line MDA-MB-231 (MB-231) and skin cancer cell A2058
were susceptible to the cytotoxic effects of H460-16-2 in
vitro.
The result of H460-16-2 cytotoxicity against MB-231 cells in
culture was further extended by its anti-tumor activity towards
these cancer cells when transplanted into mice (as disclosed in
Ser. No. 10/603,000). Pre-clinical xenograft tumor models are
considered valid predictors of therapeutic efficacy.
In the preventative in vivo model of human breast cancer, H460-16-2
treatment was significantly (p<0.0001) more effective in
suppressing tumor growth during the treatment period than an
isotype control antibody, which was identical to H460-16-2 in
structure and size but incapable of binding MB-231 cells. At the
end of the treatment phase, mice given H460-16-2 had tumors that
grew to only 1.3 percent of the control group. During the post
treatment follow-up period, the treatment effects of H460-16-2 were
sustained and the mean tumor volume in the treated groups continued
to be significantly smaller than controls until the end of the
measurement phase. Using survival as a measure of antibody
efficacy, it was estimated that the risk of dying in the H460-16-2
treatment group was about 71 percent of the antibody buffer control
group (p=0.028) at 70 days post-treatment. These data demonstrated
that H40-16-2 treatment conferred a survival benefit compared to
the control-treated groups. H460-16-2 treatment appeared safe, as
it did not induce any signs of toxicity, including reduced body
weight and clinical distress. Thus, H460-16-2 treatment was
efficacious as it both delayed tumor growth and enhanced survival
compared to the control-treated groups in a well-established model
of human breast cancer.
In addition H460-16-2 demonstrated anti-tumor activity against
MB-231 cells in an established in vivo tumor model (as outlined in
Ser. No. 10/603,000). Treatment with H460-16-2 was compared to the
standard chemotherapeutic drug, cisplatin, and it was shown that
the cisplatin and H460-16-2 treatment groups had significantly
(p<0.001) smaller mean tumor volumes compared with groups
treated with either antibody dilution buffer or the isotype control
antibody. H460-16-2 treatment mediated tumor suppression that was
approximately two-thirds that of cisplatin chemotherapy but without
the significant (19.2%) weight loss (p<0.003) and clinical
distress, including 2 treatment-associated deaths, observed with
cisplatin treatment. The anti-tumor activity of H460-16-2 and its
minimal toxicity make it an attractive anti-cancer therapeutic
agent.
In the post-treatment period, H460-16-2 showed a significant
survival benefit (p<0.02) as the risk of dying in the H460-16-2
group was about half of that in the isotype control antibody group
at >70 days after treatment. The observed survival benefit
continued past 120 days post-treatment where 100 percent of the
isotype control and cisplatin treated mice had died compared to 67
percent of the H460-16-2 treatment group. H460-16-2 maintained
tumor suppression by delaying tumor growth by 26 percent compared
to the isotype control antibody group. At 31 days post treatment,
H460-16-2 limited tumor size by reducing tumor growth by 48 percent
compared to the isotype control group, which is comparable to the
49 percent reduction observed at the end of the treatment. In the
established tumor model of breast cancer, these results indicate
the potential of H460-16-2 to maintain tumor suppression beyond the
treatment phase and demonstrates the ability of the antibody to
reduce the tumor burden and enhance survival in a mammal.
In order to validate the H460-16-2 epitope as a drug target, the
expression of H460-16-2 antigen in normal human tissues was
previously determined (Ser. No. 10/603,000). This work was extended
by comparison with an anti-CD44 antibody (clone L178). By IHC
staining with H460-16-2, the majority of the tissues again failed
to express the H460-16-2 antigen, including the cells of the vital
organs, such as the liver, kidney (except for marginal staining of
tubular epithelial cells), heart, and lung. Results from tissue
staining indicated that H460-16-2 showed restricted binding to
various cell types but had binding to infiltrating macrophages,
lymphocytes, and fibroblasts. The L178 antibody showed a similar
staining pattern. However, there were several differences of note;
staining of lymphocytes was more intense and had a wider
distribution with L178 in comparison to H460-16-2. Also, in one of
the liver samples LI 78 stained the Kupffer cells while H460-16-2
did not.
Localization of the H460-16-2 antigen and its prevalence within
breast cancer patients is important in assessing the benefits of
H460-16-2 immunotherapy to patients and designing effective
clinical trials. To address H460-16-2 antigen expression in breast
tumors from cancer patients, tumor tissue samples from 50
individual breast cancer patients were previously screened for
expression of the H460-16-2 antigen (Ser. No. 10/603,000) Current
work compared the staining of H460-16-2 to LI 78. The results of
the current study were similar to previous results and showed that
62 percent of tissue samples stained positive for the H460-16-2
antigen while 76 percent of breast tumor tissues were positive for
the L178 epitope. Expression of H460-16-2 within patient samples
appeared specific for cancer cells as staining was restricted to
malignant cells. In contrast, H460-16-2 stained 4 of 10 samples of
normal tissue from breast cancer patients while L178 stained 6.
Breast tumor expression of both the H460-16-2 and L178 antigen
appeared to be mainly localized to the cell membrane of malignant
cells, making CD44 an attractive target for therapy. H460-16-2
expression was further evaluated based on breast tumor expression
of the receptors for the hormones estrogen and progesterone, which
play an important role in the development, treatment, and prognosis
of breast tumors. No correlation was apparent between expression of
the H460-16-2 antigen and expression of the receptors for either
estrogen or progesterone. When tumors were analyzed based on their
stage, or degree to which the cancer advanced, again there was no
clear correlation between H460-16-2 antigen expression and tumor
stage. Similar results were obtained with L178.
To further extend the potential therapeutic benefit of H460-16-2,
the frequency and localization of the antigen within various human
cancer tissues was also previously determined (Ser. No.
10/603,000). Current studies compared the staining of H460-16-2 to
clone L178. The majority of these tumor types were also positive
for L178 antigen. As with human breast tumor tissue, H460-16-2 and
L178 localization occurred on the membrane of tumor cells. However,
there was substantially more membrane localization with the L178
compared to the H460-16-2 antibody. Also, of the tumor types that
were stained by both H460-16-2 and L178, 43% of the tissues showed
higher intensity staining with the L178 antibody. There appears to
be no form of CD44 that exactly matches the IHC data presented
herein based on comparisons with the IHC data from the literature.
The standard form of CD44 is normally expressed in the human brain;
H460-16-2 antigen is not. Antibodies directed against pan-CD44
isoforms do not stain the liver (including Kuppfer cells) and
positively stain the endometrial glands in all phases of the
reproductive cycle. The H460-16-2 antigen is clearly present on
Kuppfer cells and is only present on the secretory endometrial
glands of the reproductive cycle. H460-16-2 antigen is clearly
present on tissue macrophages and only the variant forms V4/5 and
V8/9 show occasional macrophage staining. The similar yet distinct
binding pattern seen with H460-16-2 in comparison to anti-CD44 L178
indicates that the H460-16-2 antigen is an unique epitope of
CD44.
As outlined herein, additional biochemical data also indicate that
the antigen recognized by H460-16-2 is one of the forms of CD44.
This is supported by studies showing that a monoclonal antibody
(L178) reactive against CD44 identifies proteins that were bound to
H460-16-2 by immunoprecipitation. Western blotting studies also
suggest that the epitope of CD44 recognized by H460-16-2 is not
present on v6 or v10. The H460-16-2 epitope is also distinguished
by being carbohydrate and conformation dependent, whereas many
anti-CD44 antibodies are directed against peptide portions of CD44.
These IHC and biochemical results demonstrate that H460-16-2 binds
to a variant of the CD44 antigen. Thus, the preponderance of
evidence shows that H460-16-2 mediate anti-cancer effects through
ligation of an unique carbohydrate dependent conformational epitope
present on a variant of CD44.
In toto, this data demonstrates that the H460-16-2 antigen is a
cancer associated antigen and is expressed in humans, and is a
pathologically relevant cancer target. Further, this data also
demonstrates the binding of the 460-16-2 antibody to human cancer
tissues and can be used appropriately for assays that can be
diagnostic, predictive of therapy, or prognostic. In addition, the
cell membrane localization of this antigen is indicative of the
cancer status of the cell due to the lack of expression of the
antigen in most non-malignant cells, and this observation permits
the use of this antigen, its gene or derivatives, its protein or
its variants to be used for assays that can be diagnostic,
predictive of therapy, or prognostic.
Other studies, involving the use of anti-CD44 antibodies, have
limitations of therapeutic potential that is not exhibited by
H460-16-2. H460-16-2 demonstrates both in vitro and in vivo
anti-tumor activity. Previously described antibodies such
MAK<CD44>M-1.1.12, MAK<CD44>M-2.42.3 and
MAK<CD44>M-4.3.16 have no in vitro or in vivo cytotoxicity
ascribed to them and VFF-18 and Mab U36 shows no intrinsic tumor
cytotoxicity. In addition other anti-CD44 antibodies that have
shown in vivo tumor effects also have certain limitations that are
not evident with H460-16-2. For example, ASML1.1, K926, anti-CD44s
and IM-78.1 show in vivo anti-tumor activity against rat, murine,
rat and murine tumors grown in xenograft models respectively.
H460-16-2 demonstrates anti-tumor activity in a model of human
cancer. H460-16-2 is also directed against human CD44 while
antibodies such as ASML 1.1 recognize only rat CD44. The clone 515
anti-CD44 antibody does inhibit peritoneal tumor implantation of a
human ovarian cell line but does not prevent or inhibit tumor
growth. H460-16-2 is capable of inhibiting human breast tumor
growth in a SCID mouse xenograft model. GKW.A3 is an anti-human
CD44 monoclonal antibody capable of inhibiting tumor growth of a
human metastasizing melanoma grown in mice in a preventative but
not an established model. H460-16-2 has demonstrated significant
anti-tumor activity in both preventative and established murine
xenograft models of human breast cancer. Consequently, it is quite
apparent that H460-16-2 has superior anti-tumor properties in
comparison to previously described anti-CD44 antibodies. It has
demonstrated both in vitro and in vivo anti-tumor activity on a
human breast tumor in SCID mice and is directed against human CD44.
It also exhibits activity in a preventative and established (more
clinically relevant) model of human breast cancer.
In all, this invention teaches the use of the H460-16-2 antigen as
a target for a therapeutic agent, that when administered can reduce
the tumor burden of a cancer expressing the antigen in a mammal,
and can also lead to a prolonged survival of the treated mammal.
This invention also teaches the use of a CDMAB (H460-16-2), and its
derivatives, to target its antigen to reduce the tumor burden of a
cancer expressing the antigen in a mammal, and to prolong the
survival of a mammal bearing tumors that express this antigen.
Furthermore, this invention also teaches the use of detecting the
H460-16-2 antigen in cancerous cells that can be useful for the
diagnosis, prediction of therapy, and prognosis of mammals bearing
tumors that express this antigen.
If a patient is refractory to the initial course of therapy or
metastases develop, the process of generating specific antibodies
to the tumor can be repeated for re-treatment. Furthermore, the
anti-cancer antibodies can be conjugated to red blood cells
obtained from that patient and re-infused for treatment of
metastases. There have been few effective treatments for metastatic
cancer and metastases usually portend a poor outcome resulting in
death. However, metastatic cancers are usually well vascularized
and the delivery of anti-cancer antibodies by red blood cells can
have the effect of concentrating the antibodies at the site of the
tumor. Even prior to metastases, most cancer cells are dependent on
the host's blood supply of their survival and anti-cancer antibody
conjugated to red blood cells can be effective against in situ
tumors as well. Alternatively, the antibodies may be conjugated to
other hematogenous cells, e.g. lymphocytes, macrophages, monocytes,
natural killer cells, etc.
There are five classes of antibodies and each is associated with a
function that is conferred by its heavy chain. It is generally
thought that cancer cell killing by naked antibodies are mediated
either through antibody-dependent cell-mediated cytotoxicity (ADCC)
or complement-dependent cytotoxicity (CDC). For example murine IgM
and IgG2a antibodies can activate human complement by binding the
C-1 component of the complement system thereby activating the
classical pathway of complement activation which can lead to tumor
lysis. For human antibodies, the most effective complement
activating antibodies are generally IgM and IgG1. Murine antibodies
of the IgG2a and IgG3 isotype are effective at recruiting cytotoxic
cells that have Fc receptors which will lead to cell killing by
monocytes, macrophages, granulocytes and certain lymphocytes. Human
antibodies of both the IgG1 and IgG3 isotype mediate ADCC.
Another possible mechanism of antibody mediated cancer killing may
be through the use of antibodies that function to catalyze the
hydrolysis of various chemical bonds in the cell membrane and its
associated glycoproteins or glycolipids, so-called catalytic
antibodies.
There are two additional mechanisms of antibody mediated cancer
cell killing which are more widely accepted. The first is the use
of antibodies as a vaccine to induce the body to produce an immune
response against the putative antigen that resides on the cancer
cell. The second is the use of antibodies to target growth
receptors and interfere with their function or to down regulate
that receptor so that effectively its function is lost.
Accordingly, it is an objective of the invention to utilize a
method for producing cancerous disease modifying antibodies from
cells derived from a particular individual which are cytotoxic with
respect to cancer cells while simultaneously being relatively
non-toxic to non-cancerous cells, in order to isolate hybridoma
cell lines and the corresponding isolated monoclonal antibodies and
antigen binding fragments thereof for which said hybridoma cell
lines are encoded.
It is an additional objective of the invention to teach methods of
utilizing the isolated monoclonal antibody or antigen binding
fragment thereof encoded by the clone deposited with the ATCC as
PTA-4621 for determining a presence of cells which express a CD44
antigenic moiety which specifically binds to an isolated monoclonal
antibody or antigen binding fragment thereof encoded by the clone
deposited with the ATCC as PTA-4621.
It is yet a further objective of the instant invention to teach
methods for enhancing the survival of a patient having a cancerous
disease via the use of an isolated monoclonal antibody or antigen
binding fragment thereof encoded by the clone deposited with the
ATCC as PTA-4621, which antibody specifically binds to a CD44
antigenic moiety.
It is an additional objective of the invention to teach CDMAB and
antigen binding fragments thereof.
It is a further objective of the instant invention to produce CDMAB
whose cytotoxicity is mediated through ADCC.
It is yet an additional objective of the instant invention to
produce CDMAB whose cytotoxicity is mediated through CDC.
It is still a further objective of the instant invention to produce
CDMAB whose cytotoxicity is a function of their ability to catalyze
hydrolysis of cellular chemical bonds.
A still further objective of the instant invention is to produce
CDMAB which are useful in a binding assay for diagnosis, prognosis,
and monitoring of cancer.
Other objects and advantages of this invention will become apparent
from the following description wherein are set forth, by way of
illustration and example, certain embodiments of this
invention.
BRIEF DESCRIPTION OF THE FIGURES
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
FIG. 1. Western blot of MDA-MB-468 membranes probed with H460-16-2.
Lane 1: Membrane proteins separated under reducing conditions. Lane
2: Membrane proteins separated under non-reducing conditions.
Molecular weight markers are indicated on the left.
FIG. 2. Western blot of membranes probed with H460-16-2. Lane 1:
MDA-MB-468 membranes. Lane 2: MDA-MB-231 membranes. Molecular
weight markers are indicated on the left.
FIG. 3. 2-Dimensional Western blot and SDS-PAGE of MDA-MB-468
membrane proteins. Panel A demonstrates the position of the 2
proteins recognized by H460-16-2. Panel B demonstrates a similar
blot probed with an isotype control antibody. Panel C shows a SYPRO
Ruby-stained gel of MDA-MB-468 membranes. Arrows indicate the
position of protein spots corresponding to panel A.
FIG. 4. 2-Dimensional Western blot of MDA-MB-231 membrane proteins.
The major binding protein is indicated with an arrow.
FIG. 5. Effect of deglycosylation on the binding of H460-16-2 to
MDA-MB-231 membranes. Panel A demonstrates the binding of H460-16-2
in a Western blot to untreated MDA-MB-231 cell membranes (Lane 2),
membranes treated with glycosidases (see text) at 37.degree. C. for
24 hr (Lane 3), and membranes treated with glycosidases at
25.degree. C. for 24 hr (Lane 4). Lane 1 shows the position of the
molecular weight markers. Panel B demonstrates the binding of the
high-mannose binding lectin GNA to a similar blot.
FIG. 6. SDS-PAGE (Panel A) and Western blot (Panel B) of MDA-MB-231
membrane proteins immunoprecipitated with H460-16-2. Lane 1: Total
MDA-MB-231 membrane proteins Lane 2: H460-16-2 immunoprecipitated
proteins. Arrow demonstrates 80 90 kD H460-16-2 binding
protein.
FIG. 7. Western blots of proteins probed with H460-16-2 (Panel A),
anti-CD44 (clone L178, Panel B) and anti-HSP90 (Panel C). Lane 1:
Total MDA-MB-231 membrane proteins; Lane 2: H460-16-2
immunoprecipitated proteins; Lane 3: Molecular weight
standards.
FIG. 8. Western blots of proteins probed with H460-16-2 (Panel A,
Lane 2), anti-CD44 (clone L178, Panel B, Lane 2), anti-CD44var6
(clone VFF-7, Panel C, Lane 2) and anti-CD44var10 (clone VFF-14,
Panel D, Lane 2). Lane 1 of each blot contains the molecular weight
standards.
FIG. 9. Representative histograms for H460-16-2 binding by FACS.
Histograms for binding of H460-16-2 (20 .mu.g/mL), 107.3 isotype
control (20 .mu.g/mL) and anti-EGFR (5 .mu.g/mL) are presented for
breast cancer (MDA-MB-231 and MDA-MB-468) and normal (Hs578.Bst and
CCD-27sk) cell lines.
FIG. 10. Representative micrographs showing the binding pattern
obtained with H460-16-2 (A) and the anti-CD44 (L178) antibody (B)
on tissues sections of tonsil from a normal human tissue array.
There is more intense and widely distributed staining of
lymphocytes with L178 than with ARH460-16-2 where the staining is
more limited to mantle zone of lymphoid nodules (black arrow),
leaving the germinal center (green arrow) with weak staining.
Magnification is 200.times..
FIG. 11. Representative micrographs showing the binding pattern
obtained with H460-16-2 (A) and the anti-CD44 (LI 78) antibody (B)
on tissues sections of liver from a normal human tissue array.
There is staining for Kupffer cells (arrows) in hepatic sinusoids
with L178 and not with H460-16-2. Magnification is 200.times..
FIG. 12. Representative micrograph of H460-16-2 binding to breast
cancer tumor (infiltrating duct carcinoma). The yellow and orange
arrows in panel point to stromal cells and sheets of malignant
cells respectively. Magnification is 100.times..
FIG. 13. Representative micrographs showing the binding pattern
obtained with H460-16-2 (A) and the anti-CD44 (L178) antibody (B)
on paget's disease breast tissue sections from a human breast
cancer tissue array. There is a membranous staining of malignant
cells (arrows) with L178 versus negative staining with H460-16-2.
Magnification is 200.times..
FIG. 14. Representative micrographs showing the binding pattern
obtained with H460-16-2 (A) and the anti-CD44 (L178) antibody (B)
on uterine cervix squamous cell carcinoma tissue sections from a
human multi-tumor tissue array. There is a stronger membranous
staining of malignant cells with H460-16-2 than with L178.
Magnification is 200.times..
FIG. 15. Representative micrographs showing the binding pattern
obtained with H460-16-2 (A) and the anti-CD44 (LI 78) antibody (B)
on adenocarcinoma lung tissue sections from a human multi-tumor
tissue array. There is +++ scoring of malignant cells (arrows) with
L178 versus +/- with H460-16-2. Magnification is 200.times..
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLE 1
Identification of Binding Proteins by Western Blotting
To identify the antigen(s) recognized by the antibody H460-16-2,
cell membranes expressing this antigen were subjected to gel
electrophoresis, and transferred to membranes. Western blotting was
used to determine proteins detected by this antibody.
1. Membrane Preparation
Previous work demonstrated binding by FACS of H460-16-2 to the
breast cancer cell lines MDA-MB-231 (MB-231) and MDA-MB-468
(MB-468). Accordingly, membrane preparations from these two cell
lines were used for antigen identification. Total cell membranes
were prepared from confluent cultures of MB-231 or MB-468 breast
cancer cells. Media was removed from flasks, and the cells were
washed 3 times with PBS. After the final wash, cells were
dissociated with Dissociation Buffer (Gibco-BRL; Grand Island,
N.Y.) for 5 min at 37.degree. C. Cells were collected and
centrifuged at 1200 rpm for 10 minutes at 4.degree. C. After
centrifugation, cell pellets were resuspended in 1 mL of hypotonic
lysis buffer containing 10 .mu.g/mL leupeptin, 10 .mu.g/mL
aprotonin and 25 .mu.g/mL 4-(2-aminoethyl)-benzenesulfonyl
fluoride. Cells were then lysed using 5 cycles of rapid freezing
and thawing. Cell lysates were centrifuged at 9500 rpm for 10 min
at 4.degree. C. to remove nuclear particulate. Supernatant was
harvested, and then centrifuged at 75,000.times.g for 57 min, at
4.degree. C. Supernatant was carefully removed from tubes, and
pellets were resuspended in 0.5 to 1 mL of hypotonic lysis buffer
containing 1 percent Triton X-100. Membranes were then assayed for
protein content, and stored at -80.degree. C.
2. 1-Dimensional SDS-PAGE
Membrane proteins were separated by 1-dimensional
SDS-polyacrylamide gel electrophoresis. 20 .mu.g of membrane
protein was loaded onto a lane of a 12 percent SDS-PAGE gel. A
sample of pre-stained molecular weight markers (Biorad;
Mississauga, ON) was run in a reference lane. The sample was
separated by electrophoresis under non-reducing conditions, in the
absence of dithiothreitol (DTT). Electrophoresis was carried out at
100 V for 10 min, followed by 65 min at 150 V. Proteins were
transferred from the gel to PVDF (Millipore; Billerica, Mass.)
membranes by electroblotting for 16 hr at 40 V. Quantitative
transfer was assessed by noting complete transfer of the prestained
markers from the gel to the membrane.
After transfer, membranes were blocked with 5 percent skim milk
powder in Tris-buffered saline containing 0.5 percent Tween (TBST)
for 2 hr. Membranes were then incubated with 2 2.5 .mu.g/mL
H460-16-2 diluted into 3 percent skim milk powder in TBST for 2 hr.
After washing 3 times with TBST, membranes were incubated with goat
anti-mouse IgG (Fc) conjugated to horseradish peroxidase (HRP) from
Jackson Immunologicals (West Grove, Pa.) for 1 hr. This incubation
was followed by washing 3 times with TBST, followed by incubation
with the HRP substrate TMB (substrate kit from Vector Laboratories;
Burlington, ON).
FIG. 1 demonstrates the results of the Western blotting as
described. H460-16-2 binds clearly to 2 molecular weight (MW)
regions of the separated MB-468 membrane proteins (Lane 2). By
comparison to the molecular weight standards, the antibody binds to
proteins of MW 80 90 kD and MW 120 150 kD. The epitope recognized
by the antibody H460-16-2 appears to be to a conformational
epitope, since the antibody was unable to bind spots transferred
from gels under reducing conditions in the presence of DTT (Lane
1). FIG. 2 compares the binding of H460-16-2 to membranes from
MB-468 (Lane 1) and MB-231 (Lane 2) cells. In MB-468 membranes,
H460-16-2 binds to both the 80 90 kD and 120 150 kD proteins with
equal intensity. In MB-231 membranes, the major binding protein is
the 80 90 kD protein, with a less intense binding protein
identified in the 120 150 kD range.
3. 2-Dimensional SDS-PAGE
In order to obtain better resolution of the binding entities, and
to further characterize the proteins, 2-dimensional electrophoresis
was carried out. Total membrane proteins (75 200 .mu.g) prepared as
described above, were precipitated using the PlusOne 2-D Clean Up
kit (Amersham; Baie D'Urfe, QC), and then resuspended in
rehydration buffer containing ampholytes in the pH range 3 10.
Samples were centrifuged to remove particulate material, and then
loaded onto IPG strips (Amersham; Baie D'Urfe, QC) in the presence
of a rehydration solution. Proteins were focussed using the
following protocol: 16 hr for rehydration; 500 V, 250 Vhrs, 1000 V,
500 Vhrs; 5000 V, 7500 V hrs. The strip was then removed from the
strip holders, and incubated in an SDS-PAGE equilibration buffer.
After 15 mm, the strip was placed on the top of an 8 percent gel,
and sealed with an agarose solution. Prestained MW markers were
loaded beside the strip. Electrophoresis was carried out at 100 V
for 10 mill, followed by 65 mill at 150 V. One of the gels was
fixed for 30 mill with 10 percent methanol/7 percent acetic acid,
and then stained with the fluorescent dye SYPRO Ruby protein gel
stain (Molecular Probes, Eugene, Oreg.). Protein spots were
visualized under UV light. From a second and third gel, proteins
were transferred from the gels to PVDF (Millipore) membranes by
electroblotting for 16 hr at 40 V. Quantitative transfer was
assessed by determining the complete transfer of the prestained
markers from the gel to the membrane.
After transfer, membranes were blocked with 5 percent skim milk
powder in Tris-buffered saline containing 0.5 percent Tween (TBST)
for 2 hr. One of the membranes was then incubated with 2 2.5
.mu.g/mL H460-16-2 diluted into 3 percent skim milk powder in TBST
for 2 hrs. A similar membrane was incubated with the same
concentration of an isotype control (Mouse anti-trinitrophenol,
IgG1,.kappa.; clone 107.3 (BD Biosciences; Oakville, ON). After
washing 3 times with TBST, membranes were incubated with goat
anti-mouse IgG (Fc) conjugated to horseradish peroxidase (HRP) from
Jackson Immunologicals (West Grove, Pa.) for 1 hr. This incubation
was followed by washing 3 times with TBST, followed by incubation
with the HRP substrate TMB (substrate kit from Vector Laboratories;
Burlington, ON).
FIG. 3a demonstrates the Western blot obtained from MB-468
membranes incubated with H460-16-2. Two distinct binding spots are
observed, with molecular weights corresponding with those obtained
by 1-dimensional electrophoresis. One is observed at a MW of
approximately 80 90 kD according to the MW standards, and is in the
acidic portion of the gel with an estimated pI of 3 4. The second
spot is in the MW range of 120 150 kD according to the MW
standards, and has a pI more basic than the 80 90 kD protein. FIG.
3b demonstrates the Western blot obtained from membranes incubated
with the isotype control antibody. No spots were visible on this
blot, indicating that the binding of H460-16-2 was not due to
non-specific binding. FIG. 3c shows a SYPRO Ruby protein gel stain
stained 2D-gel of MB-468 membrane proteins. Note that when a
similar Western blot is run with MB-23 1 membranes, only the 80 90
kD spot is observed (FIG. 4).
EXAMPLE 2
Determining Glycosylation of Antigens Bound by H460-16-2
In order to determine if the antigen(s) recognized by the antibody
H460-16-2 were glycoproteins, MB-231 membranes were incubated with
PNGase F, Endo-o-glycosidase, and sialidase A according to
manufacturer's protocol (DeglycoPro deglycosylation kit; Prozyme,
San Leandro, Calif.) for 24 hr at room temperature or at 37.degree.
C. Membranes were separated by 1-D polyacrylamide gel
electrophoresis as described, and then Western blotting was carried
out as described with H460-16-2. The results of the Western blot
are shown in FIG. 5. In MB-468 membranes that were not treated with
glycosidases, H460-16-2 recognized the expected 85-95 kD band (FIG.
5, Panel A, Lane 2). In membranes treated with glycosidases at
25.degree. C., there is a distinct shift of this band to a lower
molecular weight (Lane 4). In membranes treated with glycosidases
at 37.degree. C., the binding of 14460-16-2 is eliminated (Lane 3).
In order to determine the completeness of deglycosylation, a
similar blot was probed with the high-mannose binding lectin
galanthus nivalis agglutinin (GNA). Results observed in FIG. 5,
Panel B demonstrate that deglycosylation is incomplete at
25.degree. C. (Lane 4) and essentially complete at 37.degree. C.
(Lane 3). Thus, under conditions of complete deglycosylation,
H460-16-2 is unable to bind to its antigen.
Taken together, these results suggest that the 80-90 kD band is a
glycoprotein. In addition, these results present evidence that the
epitope recognized by H460-16-2 is carbohydrate-dependent.
EXAMPLE 3
Identification of Antigens Bound by H460-16-2
1. Immunoprecipitation
1 mL of Protein G Dynabeads (DYNAL) was washed 3 times with 0.1 M
sodium phosphate buffer, pH 6.0. 2500 .mu.g of H460-16-2 was added
to the washed beads in a total volume of 500 .mu.l. The mixture was
incubated with gentle mixing for 1 hr. Unbound antibody was removed
and the H460-16-2-coated beads were washed 3 times in 2.5 mL
volumes of 0.1 M sodium phosphate buffer, pH 7.4 containing 0.1
percent Tween-20. The H460-16-2-coated beads were washed 2 times in
5 mL of 0.2 M triethanolamine, pH 8.2 followed by the addition of 5
mL. H460-16-2 was chemically crosslinked to the beads by gentle
mixing in the presence of 5 mL of 0.2 M triethanolamine, pH 8.2
containing 20 mM dimethyl pimelimidate for 30 min. The reaction was
stopped by the addition of 5 mL of 50 mM Tris, pH 7.5. After 15 min
incubation, the H460-16-2 crosslinked beads were washed 3 times in
PBS containing 0.1 percent Tween-20. The H460-16-2-crosslinked
beads were pre-eluted by incubation with 0.1 M citrate pH 3 for 3
min followed by 3 washes in 0.1 M sodium phosphate buffer, pH 7.4
containing 0.1 percent Tween-20.
Three mg of total membrane protein from MB-23 1 cells was incubated
with H460- 16-2 chemically crosslinked beads in 0.1 M sodium
phosphate buffer, pH 7.4 containing 0.1 percent Tween-20, 5 percent
glucose, 5 percent mannose, 5 percent galactose and protease
inhibitors at 40.degree. C. for 4 hr. After incubation, the
immunoprecipate was washed 3 times in PBS containing 150 mM NaCl
and 0.1 percent Tween-20. Protein was eluted from the beads by
incubating the H460-16-2-crosslinked beads with 0.1 M citrate, pH 3
for 4 mm Eluted protein was stored at -80.degree. C.
Immunoprecipitated protein from 3 mg of protein was loaded onto a
single lane of an 8 percent non-reducing SDS-PAGE gel. A sample of
pre-stained molecular weight markers (Biorad; Mississauga, ON) was
run in a reference lane. The sample was separated by
electrophoresis at 100 V for 10mm followed by 65 mm at 150 V.
Proteins were stained with SYPRO Rubyprotein gel stain.
In a parallel Western blot, proteins immunoprecipitated as
described from 100 .mu.g of MB-231 membrane proteins were separated
by electrophoresis. Proteins were transferred from the gel to PVDF
(Millipore; Billerica, Mass.) membranes by electroblotting for 16
hr at 40 V. Quantitative transfer was assessed by noting complete
transfer of the prestained markers from the gel to the
membrane.
After transfer, the membrane was blocked with 5 percent skim milk
powder in Tris-buffered saline containing 0.5 percent Tween (TBST)
for 2 hr. The membrane was probed with 5 .mu.g/ml H460-16-2 diluted
into 3 percent skim milk powder in TBST for 2 hr. After washing 3
times with TBST, membranes were incubated with appropriate
secondary antibody: goat anti-mouse IgG (Fc) conjugated to
horseradish peroxidase (HRP) from Jackson Immunologicals (West
Grove, Pa.) for 1 hr. This incubation was followed by washing 3
times with TBST, followed by incubation with the HRP substrate TMB
(substrate kit from Vector Laboratories; Burlington, ON).
FIG. 6 depicts the gel and blot obtained from the proteins
immunoprecipitated by H460-16-2. On the gel (Panel A) there is a
band in the 80 90 kD region in the lane containing the
immunoprecipitated proteins (see arrow, Lane 2). The high molecular
weight bands are comprised of intact antibodies. No other proteins
are present in the sample. In a corresponding Western blot (Panel
B), H460-16-2 reacts strongly with the immunoprecipitated proteins
(Lane 2), with a similar binding profile to that seen in the total
membranes (Lane 1).
2. Mass Spectroscopy
The region of the gel corresponding to the 80 90 kD protein
immunoprecipitated by H460-16-2 (FIG. 6, Panel A, Lane 2) was
excised using a sterile pipette tip. This gel plug was then used
for identification of proteins by mass spectroscopy.
The sample was subjected to robotic in-gel digestion using trypsin
(ProGest) and a portion of the resulting digest supernatant was
used for MALDI/MS analysis. Spotting was performed robotically
(ProMS) with ZipTips; peptides were eluted from the C18 material
with matrix (.alpha.-cyano 4-hydroxy cinnamic acid) prepared in 60
percent acetonitrile, 0.2 percent TFA. The sample was analyzed by
nano LC/MS/MS on a Micromass Q-Tof2 using a 75 .mu.m C18 column at
a flow-rate of 200 nL/min. The MS/MS data were searched using a
local copy of MASCOT.
The proteins identified by LC/MS/MS analysis of the H460-16-2
immunoprecipitated material are presented in Table 1. A score was
assigned which is a composite score based on the number of peptides
matched, and the level of identity.
TABLE-US-00001 TABLE 1 Proteins Identified by H460-16-2
Immunoprecipitation of MDA-MB-231 Membranes Observed MW Method
Protein ID Score NCBI Accession # 80 90 KD LC/MS/MS CD44 239
gi|2134882
The only protein present in the sample was matched to human CD44,
an 80 kD glycoprotein present on the cell surface of lymphocytes
and multiple types of cancer cells.
3. Confirmation
Confirmation of the putative antigen was carried out by determining
whether a known anti-CD44 monoclonal antibody would react with the
protein immunoprecipitated by H460-16-2. Total MB-231 membrane
proteins and H460-16-2-immunoprecipitated proteins were separated
by 1-dimensional SDS-PAGE. Electrophoresis and Western blotting
were carried out as described above. Membranes were incubated with
5 10 .mu.g/ml H460-16-2, anti-CD44 (clone L178, BD Biosciences,
Oakville, ON) or anti-HSP90 (negative control; clone 16F1
(Stressgen, Victoria, BC)) diluted into 3 percent skim milk powder
in TBST for 2 hr. FIG. 7 demonstrates the results of the Western
blotting as described. Panel A shows binding of H460-16-2 to total
membrane proteins (Lane 1) and immunoprecipitated proteins (Lane
2). The major binding protein is in the 80 90 kD region, although a
band in the 120 150 kD region can also be seen in the total
membrane proteins. When a parallel blot was probed with L178 (Panel
B), a similar pattern was seen. L178 bound strongly to the protein
immunoprecipitated by H460-16-2. The binding pattern of H460-16-2
and L178 to the total membrane proteins is very similar (Panel A
and B, Lane 1). A parallel blot probed with the negative control
anti-HSP90 (Panel C) showed that this antibody did not bind to the
immunoprecipitated material, although it did bind to a distinct 90
kD protein in the total membrane preparation. This result confirms
that the binding of anti-CD44 to the immunoprecipitated protein was
specific, and not due to the presence of contaminating proteins
since HSP90 is an ubiquitous and "sticky" cellular chaperone
protein.
FIG. 8 presents the results of an experiment carried out to compare
the specificity of H460-16-2 to known anti-CD44 antibodies.
MDA-MB-231 membranes were separated by electrophoresis and
transferred to PVDF membranes as described. Membranes were probed
with H460-16-2 (Panel A), anti-CD44 (clone L178, BD Biosciences;
Panel B), anti-CD44var6 (clone VFF-7, Bender Medsystems (San Bruno,
Calif.); Panel C) and anti-CD44var10 (clone VFF-14, Bender
Medsystems; Panel D). H460-16-2 and L178 react with an 80 90 kD and
120 150 kD protein, and have identical binding patterns by Western
blot. By contrast, antibodies specific for CD44 variants 6 and 10
demonstrate differential binding from H460-16-2 and each other.
Both variant antibodies bind to a wider range of proteins, and do
not show strong binding to the 80 kD region of the gel. It is
therefore unlikely that H460-16-2 is directed towards either
variant 6 or variant 10.
The mass spectroscopic identification, as well as the confirmation
using a known antibody, demonstrates that the antigen for H460-16-2
is a form of CD44. This is also consistent with the deglycosylation
experiments in Example 2, as CD44 may contain around 50 percent
N-linked sugars by weight. These experiments also indicate that
H460-16-2 binds to a carbohydrate-dependent epitope of CD44.
EXAMPLE 4
The hybridoma cell line H460-16-2 was deposited, in accordance with
the Budapest Treaty, with the American Type Culture Collection,
10801 University Blvd., Manassas, Va. 20110-2209 on Sep. 4, 2002,
under Accession Number PTA-4621. In accordance with 37 CFR 1.808,
the depositors assure that all restrictions imposed on the
availability to the public of the deposited materials will be
irrevocably removed upon the granting of a patent.
Antibody Production
H460-16-2 monoclonal antibody was produced by culturing the
hybridomas in CL-1000 flasks (BD Biosciences, Oakville, ON) with
collections and reseeding occurring twice/week and standard
antibody purification procedures with Protein G Sepharose 4 Fast
Flow (Amersham Biosciences, Baie d'Urfe, QC). It is within the
scope of this invention to utilize monoclonal antibodies that are
human, humanized, chimerized or murine antibodies. H460-16-2 was
compared to a number of both positive (anti-Fas (EOS9.1, IgM,
kappa, 10 .mu.g/mL, eBioscience; San Diego, Calif.), anti-Her2/neu
(IgG1, kappa, 10 .mu.g/mL, Inter Medico; Markham, ON), anti-EGFR
(C225, IgG1, kappa, 5 .mu.g/mL, Cedarlane; Hornby, ON),
Cycloheximide (100 ptM, Sigma; Oakville, ON), NaN.sub.3 (0.1
percent, Sigma; Oakville, ON)) and negative (107.3 (anti-TNP, IgG1,
kappa, 20 .mu.g/mL, BD Biosciences; Oakville, ON), G155-178
(anti-TNP, IgG2a, kappa, 20 .mu.g/mL, BD Biosciences; Oakville,
ON), MPC-11 (antigenic specificity unknown, IgG2b, kappa, 20
.mu.g/mL, BD Biosciences; Oakville, ON), J606 (anti-fructosan,
IgG3, kappa, 20 .mu.g/mL, BD Biosciences; Oakville, ON), IgG Buffer
(2 percent)) controls in a cytotoxicity assay (Table 3). Breast
cancer (MB-231, MB-468), melanoma (A2058, A375), colon cancer
(HT-29), lung cancer (NCI-H460, A549), ovarian cancer (OVCAR-3),
prostate cancer (PC-3), and non-cancer (CCD-27sk, Hs578.Bst,
Hs888.Lu) cell lines were tested (all from the ATCC, Manassas,
Va.). The Live/Dead cytotoxicity assay was obtained from Molecular
Probes (Eugene, Oreg.). The assays were performed according to the
manufacturer's instructions with the changes outlined below. Cells
were plated before the assay at the predetermined appropriate
density. After 2 days, 100 .mu.L of purified antibody was diluted
into media, and then were transferred to the cell plates and
incubated in a 5 percent CO.sub.2 incubator for 5 days. The plate
was then emptied by inverting and blotted dry. Room temperature
DPBS containing MgCl.sub.2 and CaCl.sub.2 was dispensed into each
well from a multichannel squeeze bottle, tapped 3 times, emptied by
inversion and then blotted dry. 50 .mu.L of the fluorescent
Live/Dead dye diluted in DPBS containing MgCl.sub.2 and CaCl.sub.2
was added to each well and incubated at 37.degree. C. in a 5
percent CO.sub.2 incubator for 30 min. The plates were read in a
Perkin-Elmer HTS7000 fluorescence plate reader and the data was
analyzed in Microsoft Excel and the results were tabulated in Table
2. The data represented an average of 4 experiments tested in
triplicate and presented qualitatively in the following fashion:
4/4 experiments greater than threshold cytotoxicity (+++), 3/4
experiments greater than threshold cytotoxicity (++), 2/4
experiments greater than threshold cytotoxicity (+). Unmarked cells
in Table 2 represented inconsistent or effects less than the
threshold cytotoxicity. The H460-16-2 antibody produced selective
cytotoxicity in A2058 melanoma cells and MB-231 breast cancer cells
but did not produce cytotoxicity against the remaining cancer
cells, demonstrating properties of specific cytotoxicity towards
cancer cells. Importantly the isolated antibody did not produce
cytotoxicity against a number of non-cancer cells such as CCD-27sk,
Hs578.Bst or Hs888.Lu. The chemical cytotoxic agents induced their
expected cytotoxicity while a number of other antibodies which were
included for comparison also performed as expected given the
limitations of biological cell assays.
TABLE-US-00002 TABLE 2 In Vitro Cytotoxicity Normal Melanoma Breast
Lung Colon Ovary Prostate CCD- A2058 A375 MB-231 MB-468 NCl-H460
A549 HT-29 OVCAR-3 PC-3 27sk t Hs888.Lu H460-16-2 (20 .mu.g/mL) + +
Positive Anti-Fas ++ +++ +++ Controls (20 .mu.g/mL) Anti-Her2/neu
++ (10 .mu.g/mL) Anti-EGFR + (c528, 5 .mu.g/mL) Cycloheximide (100
.mu.M) +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ + +++ Nagative IgG1
(107.3, 20 .mu.g/mL) Controls Human IgG (10 .mu.g/mL) + IgG Buffer
(2%)
Cells were prepared for FACS by initially washing the cell
monolayer with DPBS (without Ca.sup.++ and Mg.sup.++). Cell
dissociation buffer (INVITROGEN; Burlington, ON) was then used to
dislodge the cells from their cell culture plates at 37.degree. C.
After centrifugation and collection the cells were resuspended in
DPBS containing MgCl.sub.2, CaCl.sub.2 and 25 percent fetal bovine
serum at 4.degree. C. (wash media) and counted, aliquoted to
appropriate cell density, spun down to pellet the cells and
resuspended in staining media (DPBS containing MgCl.sub.2,
CaCl.sub.2 and 2 percent fetal bovine serum) at 4.degree. C. in the
presence of test antibodies (H460-16-2) or control antibodies
(isotype control, anti-Her2/neu or anti-EGFR) at 20 .mu.g/mL on ice
for 30 minutes. Prior to the addition of Alexa Fluor 488-conjugated
secondary antibody the cells were washed once with wash media. The
Alexa Fluor 488-conjugated antibody in staining media was then
added for 20 min. The cells were then washed for the final time and
resuspended in staining media containing 1 .mu.g/mL propidium
iodide. Flow cytometric acquisition of the cells was assessed by
running samples on a FACScan using the CellQuest software (BD
Biosciences; Oakville, ON). The forward (FSC) and side scatter
(SSC) of the cells were set by adjusting the voltage and amplitude
gains on the FSC and SSC detectors. The detectors for the three
fluorescence channels (FL1, FL2, and FL3) were adjusted by running
cells stained with purified isotype control antibody followed by
Alexa Fluor 488-conjugated secondary antibody such that cells had a
uniform speak with a median fluorescent intensity of approximately
1 5 units. Live cells were acquired by gating for FSC and propidium
iodide exclusion. For each sample, approximately 10,000 live cells
were acquired for analysis and the results presented in Table
3.
Table 3 tabulated the mean fluorescence intensity fold increase
above isotype control and is presented qualitatively as: less than
5 (-); 5 to 50 (+); 50 to 100 (++); above
TABLE-US-00003 TABLE 3 FACS Analysis CCD- MB- Hs578. Antibody
NCl-H460 A549 Hs888.Lu HT-29 PC-3 OVCAR-3 A375 A2058 27sk MB-231 -
468 Bst Anti-EGFR +(100%) +(100%) ++ ++ +(98%) ++ +(99%) - +(98%)
+++ +++ +(97%) Anti-HER2/neu - - - +(32%) - +(43%) +(67%) +(31%) -
+(22%) - - Anti-Fas - - +(48%) +(5%) - - - - +(9%) - - +(4%)
H460-16-2 +(100%) ++ +++ ++ ++ +(33%) +++ +++ ++ +++ +++ +++
100 (E+++) and in parenthesis, the percentage of cells stained.
Representative histograms of H460-16-2 antibodies were compiled for
FIG. 9 and evidence the binding characteristics, inclusive of
illustrated bimodal peaks in some cases. H460-16-2 bound 100 fold
above isotype control to a number of cancer cell types including
melanoma and breast cancer cells; 5 to 100 fold to lung, colon,
prostate, and ovarian cancer cells. There was binding of H460-16-2
antibodies to non-cancer cells, however that binding did not
produce cytotoxicity. This was evidence that binding was not
necessarily predictive of the outcome of antibody ligation of its
cognate antigen, and was a non-obvious finding. This suggested that
the context of antibody ligation in different cells was
determinative of cytoxicity rather than just antibody binding.
EXAMPLE 5
Normal Human Tissue Staining
IHC studies were previously conducted to characterize H460-16-2
antigen distribution in humans (Ser. No. 10/603,000). The current
studies compare H460-16-2 to an antibody directed against CD44
(L178) since the H460-16-2 antigen may be a cancer variant of the
standard form of CD44 as determined previously by biochemical
methods. Binding of antibodies to 59 normal human tissues was
performed using a human, normal organ tissue array (Imgenex, San
Diego, Calif.). All primary antibodies (H460-16-2; L178 anti-CD44
(also known as anti-HCAM, BD PharMingen, Oakville, ON); and mouse
IgG.sub.1 negative control (Dako, Toronto, ON)) were diluted in
antibody dilution buffer (Dako, Toronto, ON) to a concentration of
5 .mu.g/ml (found to be the optimal concentration in previous
optimization steps). The negative control antibody has been shown
to be negative to all mammalian tissues by the manufacturer. The
procedure for IHC is as follows.
Tissue sections were deparaffinized by drying in an oven at
58.degree. C. for 1 hour and dewaxed by immersing in xylene 5 times
for 4 minutes each in Coplin jars. Following treatment through a
series of graded ethanol washes (100% 75%) the sections were
re-hydrated in water. The slides were immersed in 10 mM citrate
buffer at pH 6 (Dako, Toronto, Ontario) then microwaved at high,
medium, and low power settings for 5 minutes each and finally
immersed in cold PBS. Slides were then immersed in 3% hydrogen
peroxide solution for 6 minutes, washed with PBS three times for 5
minutes each, dried, incubated with Universal blocking solution
(Dako, Toronto, Ontario) for 5 minutes at room temperature.
H460-16-2, L178 or isotype control antibody (directed towards
Aspergillus niger glucose oxidase, an enzyme which is neither
present nor inducible in mammalian tissues) were diluted in
antibody dilution buffer (Dako, Toronto, Ontario) to its working
concentration (5 .mu.g/mL for each antibody) and incubated for 1
hour at room temperature. The slides were washed with PBS 3 times
for 5 minutes each. Immunoreactivity of the primary antibodies was
detected/visualized with HRP conjugated secondary antibodies as
supplied (Dako Envision System, Toronto, Ontario) for 30 minutes at
room temperature. Following this step the slides were washed with
PBS 3 times for 5 minutes each and a color reaction developed by
adding DAB (3,3'-diaminobenzidine tetrahydrachloride, Dako,
Toronto, Ontario) chromogen substrate solution for immunoperoxidase
staining for 10 minutes at room temperature. Washing the slides in
tap water terminated the chromogenic reaction. Following
counterstaining with Meyer's Hematoxylin (Sigma Diagnostics,
Oakville, ON), the slides were dehyrdated with graded ethanols (75
100%) and cleared with xylene. Using mounting media (Dako
Faramount, Toronto, Ontario) the slides were coverslipped. Slides
were microscopically examined using an Axiovert 200 (Zeiss Canada,
Toronto, ON) and digital images acquired and stored using Northern
Eclipse Imaging Software (Mississauga, ON). Results were read,
scored and interpreted by a pathologist.
Table 4 presents a summary of the results of H460-16-2 and L178
anti-CD44 staining of an array of normal human tissues. The
staining of tissues with H460-16-2 is similar to that described
previously (Ser. No. 10/603,000). It should be again noted that the
antigen is generally not present on cells in the vital organs,
including the liver, kidney, heart and lung. The H460-16-2antibody
does bind to macrophages and lymphocytes, and their presence is
observed in some of the organs in these sections. However, there
was a wider distribution and a higher intensity of staining of
lymphocytes seen with the L178 anti-CD44 antibody (FIG. 10).
Tissues that were positive for H460-16-2 were also usually positive
for L178 anti-CD44 (sometimes to a greater intensity). Tissues that
were negative for H460-16-2 were also generally negative for L178
anti-CD44 albeit there are a few exceptions such as one sample of
liver (FIG. 11) and esophagus. These results demonstrate that
H460-16-2 binds to a slightly smaller subset of the tissues
recognized by the L178 anti-CD44 antibody and within tissues the
intensity of staining is also sometimes less. These results show
that the antigen for H460-16-2 is not widely expressed on normal
tissues, and that the antibody binds specifically to a limited
number of tissues in humans. It also supports the biochemical data
in that H460-16-2 is directed against an epitope of CD44, albeit to
a slightly different variant than the one recognized by the L178
used for these IHC studies.
TABLE-US-00004 TABLE 4 Comparison of L178 anti-CD44 and H460-16-2
IHC on Human Normal Tissue Sec. Isotype No. Organ Anti-CD44
H460-15-2 control 1 *Skin +++ Keratinocytes of all epidermal
layers, Fibrolasts, ++ Keratinocytes of all epidermal layers,
Fibrolasts - SMF of blood vessels 2 *Skin +++ Keratinocytes of all
epidermal layers, Fibrolasts +++ Keratinocytes of all epidermal
layers, Fibrolasts - 3 Subcutis fat - - - 4 Breast +/- Ductular
epithelium, Myoepithelium & Fibroblasts + Myoepithelium - 5
Breast +++ Myoepithelium & Fibroblasts ++ Myoepithelium &
Fibroblasts - 6 Spleen +++ Lymphocytes predominantly in the
periarteriolar area ++ Lymphocytes predominantly in the
periarteriolar area - 7 Spleen +++ Lymphocytes predominantly in the
periarteriolar area ++ Lymphocytes predominantly in the
periarteriolar area - 8 Lymphnode +++ Lymphocytes + Lymphocytes - 9
Lymphnode - - - 10 Skeletal muscle +/- Blood vessels +/- Blood
vessels - 11 Nasal Mucosa - NR CS CS 12 Lung +++ Macrophages &
Fibroblasts ++ Macrophages - 13 Lung +++ Pneumocytes, Lymphocytes
& Macrophages +++ Lymphocytes & Macrophages - 14 Bronchus -
NR - NR - NR 15 Heart **- **- *- 16 Salivary gland +++ Acinar &
Doctal epithelium, + Peripheral nerve ++ Acinar & Doctal
epithelium, + Peripheral nerve - 17 Liver +++ Kupffer cells +++
Kupffer cells - 18 Liver +++ Kupffer cells +++ Kupffer cells - 19
Liver ++ Kupffer cells - - 20 Gall bladder +++ Mucosal epithelium,
Lymphocytes, Fibroblasts & SMF + Mucosal epithelium, +++
Lymphocytes - 21 Pancreas + Acinar epithelium + Acinar epithelium -
22 Pancreas +++ Acinar epithelium ++ Acinar epithelium - 23 Tonsil
+++ Lymphocytes all over the lymphatic nodules ++ Lymphocytes
mainly in mantle zone - 24 Esophagus + Squamous epithelium in basal
layers, Fibroblasts - - 25 Esophagus +++ Squamous epithelium in
basal layers & Fibroblasts +++ Squamous epithelium in basal
layers & Fibroblasts - 26 ***Stomach ++ Gastric gland
epithelium in basal parts +++ Gastric gland epithelium in basal
parts of the - body of the glands & Lymphocytes glands, +++
Lymphocytes 27 ***Stomach +++ Gastric gland epithelium in basal
parts +/- Gastric gland epithelium in basal parts of the - body of
the glands & Lymphocytes glands, +++ Lymphocytes 28 Stomach +++
Lymphocytes & Fibroblasts +++ Lymphocytes & Fibroblasts -
antrum 29 Stomach ++ Blood vessels & Fibroblasts ++ Blood
vessels & Fibroblasts - Smooth muscle 30 Duodenum +++ Glandular
epithelium in deeper part +/- Glandular epithelium in deeper part
of the - of the glands & Lymphocytes glands, ++ Lymphocytes 31
Small bowel +/- Glandular epithelium in deeper part of the ++
Lymphocytes in lamina propria - glands, +++ Lymphocytes in lamina
propria 32 Small bowel +++ Lymphocytes mainly in mantle zone of +++
Lymphocytes mainly in mantle zone of - lymphatic modules lymphatic
nodules 33 Appendix ++ Glandular epithelium, +++ Lymphocytes +/-
Mucosal epithelium, +++ Lymphocytes - 34 Colon +/- Glandular
epithelium, +++ Lymphocytes, SMF & +++ Lymphocytes - Ganglion
cells 35 Colon +++ Lymphocytes, Ganglion cells & Endothelium
+++ Lymphocyte of lamina propria & Lymphatic - of blood vessels
nodules' mantle zone 36 Rectum +++ Lymphocytes & Fibroblasts
+++ Lymphocytes & - Fibroblasts 37 Kidney cortex ++ Endothelium
of blood vessels & Interstitial Fibroblasts +/- Inteertinal
fibroblasts - 38 Kidney cortex +/- Tubular epithelium &
Intestitial fibroblasts +/- Tubular epithelium & Intestitial
fibroblasts - 39 Kidney + SMF & Fibroblasts + SMF &
Fibroblasts - Medulla 40 Urinary +++ Superficial transitional
epithelium, Lymphocytes & +/- Transitional Epithelium, ++
Lymphocytes & - Bladder Macrophages Macrophages 41 Prostate +++
Myoepithelium +/- Glandular epithelium, +++ Myoepithelium - 42
Prostate +++ Myoepithelium & Fibroblasts ++ Myoepithelium - 43
Seminal vesicle +/- Mucosal epithelium, ++ Outer longitudinal +/-
Mucosal epithelium, ++ Outer longitudinal SMF of - SMF of musculars
externs musculars externs 44 Testis +/- Intestitial bloos vessels
& Fibroblasts +/- Intestitial fibroblasts - 45 Endometrium +++
Stromal fibroblasts ++ Stromal fibroblasts - profilarative 46
Endometrium +++ Glandular epithelum & Stroma +++ Glandular
epithelum & Stroma - secretory 47 Myometrium +++ SMF &
Fibroblasts +++ SMF - 48 Uterine cervix +++ Squamous epithelium is
deeper layers & Fibroblasts +++ Squamous epithelium in deeper
layers & Fibroblasts - 49 Salpinx + Fibroblasts & SMF +
Fibroblasts & SMF - 50 ***Ovary +/- SMF of blood vessels +/-
SMF of blood vessels - 51 Placenta, villi ++ Endothelium of blood
vessels ++ Endothelium of blood vessels - 52 Placenta, villi ++
Endothelium of blood vessels +/- Fibroblasts - 53 Umbilical cord -
- - 54 Adrenal gland **+/- **+/- **+/- 55 Thyroid +/- Blood vessels
& Parafollicular cells +/- Blood vessels & Parafollicular
cells - 56 Thymus + Lymphocytes +/- Lymphocytes - PS 57 Brain white
- - - matter 58 Brain gray - - - matter 59 Cerebellum - - -
Abbrevations: *Original pigmented stratum basale, **Endogenous
cytoplasmic pigment/back ground staining, ***Stomach antrum (not
stomach boby), ****Ovarian stroma only, SMF: Smooth muscle fibers,
NR: The section is not representative, CS: The section is
completely sloughed, PS: the section is partially sloughed.
EXAMPLE 6
Human Breast Tumor Tissue Staining
A previous IHC study was undertaken to determine the cancer
association of the H460-16-2 antigen with human breast cancers and
whether the H460-16-2 antibody was likely to recognize human
cancers (Ser. No. 10/603,000). Currently, a comparison was made for
L178 anti-CD44 staining, and an antibody directed towards
Aspergillus niger glucose oxidase, an enzyme which is neither
present nor inducible in mammalian tissues (negative control). A
breast cancer tissue array derived from 50 breast cancer patients
and 9 samples derived from non-neoplastic breast tissue in breast
cancer patients was used (Imgenex Corporation, San Diego, Calif.).
The following information was provided for each patient: age, sex,
American Joint Committee on Cancer (AJCC) tumor stage, lymph node,
estrogen receptor (ER) and projesterone receptor (PR) status. The
procedure for IHC from Example 5 was followed. All antibodies were
used at a working concentration of 5 .mu.g/ml.
Tables 5 and 6 provide summaries of H460-16-2 and L178 anti-CD44
antibody staining of breast cancer tissue arrays respectively. Each
array contained tumor samples from 50 individual patients. Overall,
62 percent of the 50 patients tested were positive for H460-16-2
antigen compared to 76 percent for CD44. In cases where both
H460-16-2 and L178 anti-CD44 stained the same tissue, 43% of the
samples had higher intensity staining with the L178 anti-CD44 in
comparison to H460-16-2. For both the H460-16-2 and CD44 antigen,
only 4 and 6 out of 10 normal breast tissue samples from breast
cancer patients were positive respectively. No clear correlation
between estrogen and progesterone receptor status was evident. It
also did not appear to be a trend to greater positive expression of
the H460-16-2 and CD44 antigen with higher tumor stage.
TABLE-US-00005 TABLE 5 Human Breast Tumor IHC Summary for H460-16-2
Binding Score Total # - +/- + ++ +++ Total Positive % Positive
Patient Samples Tumor 50 19 19 4 3 5 31 62 Normal 10 0 1 0 2 1 4 40
ER Status ER+ 28 13 13 1 1 0 15 54 ER- 22 6 8 3 0 5 16 73 Unknown 0
0 0 0 0 0 0 0 PR Status PR+ 19 9 8 1 1 0 10 53 PR- 30 8 14 3 0 5 22
73 Unknown 1 0 1 0 0 0 1 100 AJCCTumorStage T1 4 2 1 1 0 0 2 50 T2
21 6 9 1 1 4 15 71 T3 20 9 9 1 0 1 11 55 T4 5 2 2 1 0 0 3 60
TABLE-US-00006 TABLE 6 Human Breast Tumor IHC Summary for Anti-CD44
(L178) Total # - +/- + ++ +++ Total Positive % Positive Patient
Samples Tumor 50 12 20 3 7 8 39 78 Normal 10 4 1 0 3 2 6 60 ER
Status ER+ 28 7 14 1 4 2 21 75 ER- 22 5 6 2 3 6 17 77 Unknown 0 0 0
0 0 0 0 0 PR Status PR+ 19 5 9 1 2 2 14 74 PR- 30 7 10 2 5 6 23 77
Unknown 1 0 1 0 0 0 1 100 AJCCTumorStage T1 4 1 2 0 0 1 3 75 T2 21
5 6 2 2 6 16 76 T3 20 6 9 1 3 1 14 70 T4 5 0 3 0 2 0 5 100
The H460-16-2 staining was specific for cancerous cells in
comparison to normal cells as demonstrated in FIG. 12 where stromal
cells were clearly negative and sheets of malignant cells were
highly positive. The cellular localization pattern seen with the
H460-16-2 antigen was confined to the cell membrane in the majority
of cases. The L178 CD44 antibody stained more breast cancer samples
and showed a higher degree of membrane than cytoplasmic
localization compared to H460-16-2 (Table 7). L178 anti-CD44 also
stained malignant cells of Paget's disease, which was not the case
for H460-16-2 (FIG. 13). L178 anti-CD44 stained the same samples of
normal tissue from breast cancer patients as H460-16-2 (plus 1 that
was partially sloughed for the H460-16-2 sample). These results
suggest the antigen for H460-16-2 may be expressed by almost two
thirds of breast cancer patients. The staining pattern showed that
in patient samples, the antibody is highly specific for malignant
cells and the H460-16-2 antigen is localized to the cell membrane
thereby making it an attractive drugable target. The similar albeit
more limited staining of H460-16-2 versus L178 anti-CD44 antibody
again demonstrates the likelihood of the H460-16-2 epitope being a
more restricted variant of CD44.
TABLE-US-00007 TABLE 7 Comparison of L178 anti-CD44 and H460-16-2
IHC on Human Tumor and Normal Breast Tissue Sec. Isotype No. Sex
Age Diagnosis Anti-CD44 H460-46-2 Control 1 F 28 Infiltrating
ductal carcinoma +/- +/- - 2 F 71 Solid papillary carcinoma ++ M
+/- - 3 F 26 Infiltrating ductal carcinoma +/- - - 4 F 43
Infiltrating ductal carcinoma +/- - - 5 F 39 Infiltrating ductal
carcinoma +/- +/- Tumor & Necrotic area - 6 F 46 Ductal
carcinoma in situ +/- - - 7 F 47 Infiltrating ductal carcinoma +++
M +++ M - 8 M 67 Infiltrating ductal carcinoma - Tumor, + Stroma
only - - 9 F 33 Infiltrating ductal carcinoma - Tumor, + Stroma
only - Tumor, ++ Stroma - 10 F 47 Infiltrating ductal carcinoma +/-
+/- - 11 F 49 Invasive lobular carcinoma - Tumor, ++ Infiltrating
lymphocytes - - 12 F 46 Infiltrating ductal carcinoma ++ M +/- - 13
F 39 Infiltrating ductal carcinoma +/- - - 14 F 43 Infiltrating
lobular carcinoma + MC +/- - 15 F 54 Infiltrating lobular carcinoma
- Tumor, + Stroma +/- - 16 F 58 Infiltrating ductal carcinoma ++ M
Tumor, +++ Stroma + MCTumor, ++ Necrotic area - 17 F 37
Infiltrating ductal carcinoma - Tumor, + Stroma +/- - 18 F 43
Infiltrating ductal carcinoma ++ M Tumor, +++ Stroma + MC Tumor,
+++ Stroma - 19 F 51 Infiltrating ductal carcinoma +++ M ++ M - 20
F 80 Medullar carcinoma +++ M +++ MC - 21 F 36 Infiltrating ductal
carcinoma +++ M +++ M - 22 F 59 Infiltrating ductal carcinoma +/-
Tumor, ++ Stroma +/- Tumor, ++ Stroma - 23 F 34 Ductal carcinoma in
situ +/- +/- Tumor & Nerotic area - 24 F 54 Infiltrating ductal
carcinoma +/- +/- - 25 F 47 Infiltrating ductal carcinoma +++ M +/-
- 26 F 53 Infiltrating ductal carcinoma + Tumor, +++ Stroma -
Tumor, ++ Stroma - 27 F 59 Infiltrating ductal carcinoma +/- Tumor,
+++ Lymphocytes +/- Tumor, +++ Lymphocytes - 28 F 60 Signet ring
cell carcinoma - Tumor, +/- Stroma - - 29 F 37 Infiltrating ductal
carcinoma +/- +/- - 30 F 46 Infiltrating ductal carcinoma ++ Tumor,
+++ Infiltrating Lymphocytes +/- Tumor & Stroma - 31 F 35
Infiltrating ductal carcinoma +/- - - 32 F 47 Infiltrating ductal
carcinoma - Tumor, ++ Necrotic cells - Tumor, +/- Necrotic cells -
33 F 54 Infiltrating ductal carcinoma +/- Tumor & Stroma - - 34
F 47 Infiltrating ductal carcinoma +++ M +++ M - 35 F 41
Infiltrating ductal carcinoma - - - 36 F 38 Infiltrating ductal
carcinoma ++ M +/- - 37 F 55 Infiltrating ductal carcinoma - Tumor,
+/- Stroma - - 38 M 65 Infiltrating ductal carcinoma - Tumor, ++
Stroma +/- Stroma - 39 M 66 Infiltrating ductal carcinoma - - - 40
F 44 Infiltrating ductal carcinoma +/- - Tumor, + Infiltrating
Lymphocytes - 41 F 52 Metastatic carcinoma in lymph node +/- Tumor,
+++ Infiltrating lymphocytes +/- Tumor & Stroma - 42 F 32
Metastatic carcinoma in lymph node +/- - - 43 F 58 Metastatic
carcinoma in lymph node ++ M + MC - 44 F 52 Metastatic carcinoma in
lymph node - - - 45 F 58 Metastatic carcinoma in lymph node +/- +/-
- 46 F 38 Metastatic carcinoma in lymph node +/- Tumor, +++
Infiltrating lymphocytes - Tumor, + Lymphocytes - 47 F 45
Metastatic carcinoma in lymph node +++ M + MC - 48 F 45 Metastatic
carcinoma in lymph node + M +/- - 49 F 29 Metastatic carcinoma in
lymph node +++ M +++ M - 50 F 61 Metastatic carcinoma in lymph node
+/- Tumor, ++ Stroma +/- Tumor, ++ Stroma - *51 F 46 Nipple +++
Keratinocytes ++ Keratinocytes - *52 F 47 Nipple +/- Tumor cells -
- *53 F 40 Normal breast - - - *54 F 43 Normal breast ++
Myoepithelium +++ Myoepithelium - *55 F 40 Normal breast ++
Myoepithelium ++ Myoepithelium - *56 F 40 Normal breast +++
Myoepithelium & Fibroblasts +/- Myoepithelium & Fibroblasts
- *57 F 45 Normal breast - - - *58 F 44 Normal breast - - - *59 F
37 Normal breast - - - 60 F 51 Normal breast ++ Myoepithelium &
Fibroblasts - PS - Abbreviations: *Non-neoplastic breast tissue in
breast cancer patient, PS: The section is partially sloughed, M:
Membrane staining, C: Cytoplasmic staining.
EXAMPLE 7
Human Tumor Tissue Staining
To determine whether the H460-16-2 antigen is expressed on other
human cancer tissues in addition to breast cancer, H460-16-2 was
previously used on a multiple human tumor tissue array (Ser. No.
10/603,000; Imgenex, San Diego, Calif.). In furthering those
studies, the staining pattern of H460-16-2 was compared to that of
L178 anti-CD44 The following information was provided for each
patient: age, sex, organ and diagnosis. The staining procedure used
was the same as the one outlined in Example 5. The same negative
control antibody was used as described for the human breast tumor
tissue array. All antibodies were used at a working concentration
of 5 .mu.g/mL.
As outlined in Table 8, H460-16-2 stained a number of various human
cancers besides breast with results consistent with those described
previously (Ser. No. 10/603,000). As seen with the breast cancers,
H460-16-2 staining was localized on the membrane (FIG. 14) and in
some of the cancers also within the cytoplasm of cancerous cells.
L178 anti-CD44 antibody had greater membrane versus cytoplasmic
staining with again a higher percentage of tumor tissues staining
positive and with greater intensity than that observed with
H460-16-2 (FIG. 15).
TABLE-US-00008 TABLE 8 Comparison of L178 anti-CD44 and H460-16-2
IHC on Multiple Human Tumor Types Sec. Isotype No. Age Sex Organ
Diagnosis Anti-CD44 H460-16-2 Control 1 59 M Skin Malignant
Melanoma +++ M +++ M - 2 25 F Skin SSC +/- - - 3 50 F Breast
Infiltrating ductal carcinoma +++ Tumor & Stroma + Tumor, +++
Stroma - 4 57 F Breast Invasive papillary carcinoma +/- Tumor &
Stroma +/- Tumor & Stroma - 5 35 F Breast Infiltrating lobular
carcinoma + M +/- - 6 40 M Lymph node Malignant lymphoma,
Immunoplastic +++ M +++ M - 7 58 M Lymph node Metastatic
adenocarcinoma +++ M +/- - from stomach 8 53 F Bone Osteosarcoma
+++ M + M/C - 9 26 M Bone Giant cell tumor ++ M + M/C - 10 40 M
bone Chondro-sarcoma CS CS CS 11 51 F Soft tissue Liposarcoma - - -
12 47 F Soft tissue Neuro-fibromatosis ++ M/C + M/C - 13 74 M Nasal
cavity Inverted papilloma +++ M ++ M - 14 57 M Larynx SCC +++ M +++
M - 15 60 M Lung Adenocarcinoma +++ M +/- - 16 51 F Lung SCC +++ M
+++ M/C - 17 68 F Lung Adenocarcinoma ++ M +/- - 18 60 M Lung Small
cell carcinoma +/- Necrotic area +/- - 19 88 F Tongue SCC +++ M +++
M -* 20 34 F Parotid gland Pleomorphic adenoma ++ M/C - - 21 50 F
Parotid gland Warthin tumor +++ MC +++ M/C - 22 40 F Parotid gland
Pleomorphic adenoma +++ M ++ M/C - 23 56 M Submandibular Salivary
duct carcinoma +/- - - gland 24 69 F Liver Cholangio-carcinoma +/-
Tumor, ++ Stroma +/- - 25 51 M Liver Metastatic gastric carcinoma -
Tumor, +++ Necrotic area - - 26 64 M Liver HCC +/- Tumor, +/-
Tumor, - ++ Extracellular secretion ++ Extracellular secretion 27
62 F Gall bladder Adenocarcinoma +++ Tumor ++ Tumor &
Lymphocytes - 28 64 F Pancreas Adenocarcinoma ++ M ++ M/C - 29 68 M
Esophagus SCC ++ M +/- - 30 73 M Stomach Adenocarcinoma, Poorly +/-
Tumor, ++ Stroma + M/C Tumor & Stroma - differentiated 31 63 M
Stomach Adenocarcinoma, Moderatly +/- Tumor, +++ Infiltrating ++
M/C - differentiated lymphocytes 32 59 F Stomach Signet ring cell
carcinoma +++ M ++ M/C - 33 62 M Stomach Malignant lymphoma +++ M
+++ M/C -* 34 51 M Stomach Borderline stromal tumor +/- Stroma - -
35 42 M Small Intestine Malignant stromal tumor ++ M - - 36 52 F
Appendix Pseuomynomapentonia PS, +++ Infiltrating lymphocytes - -
37 53 M Colon Adenocarcinoma + M/C Tumor & Stroma + M/C - 38 67
M Rectum Adenocarcinoma +/- ++ M - 39 75 F Kidney Transitional cell
carcinoma ++ M + M/C - 40 54 F Kidney Renal cell carcinoma +++ M
+/- - 41 75 F Kidney Renal cell carcinoma +/- +/- - 42 65 M Urinary
bladder Poorly differentisted carcinoma +++ Infiltrating
lymphocytes ++ M/C - 43 67 M Urinary bladder Transitional cell
carcinoma, +/- Stroma - - High grade 44 62 M Prostate
Adenocarcinoma +++ M +++ M -* 45 30 M Testis Seminoma +/- Tumor,
+++ Stroma +/- - 46 68 F Uterus Endometrial adenocarcinoma ++
Stroma mainly ++ Stroma mainly - 47 57 F uterus Leimyosacoma ++ C +
PS - 48 45 F uterus Leiomyoma ++ C + C - 49 63 F Uterine cervix SCC
+++ M +++ M - 50 12 F Ovary Endodermal sinus tumor +/- - - 51 33 F
Ovary Mucinous adenocarcinoma + M/C - - 52 70 F Ovary Fibrothecoma
- - - 53 67 F Adrenal gland Corticalcarcinoma - - -* 54 61 F
Adrenal gland Pheohromcytoma - - - 55 54 M Thyroid Papillary
carcinoma ++ M ++ M/C - 56 58 F Thyroid Minimally invasive
follicular +++ M ++ M - carcinoma 57 74 M Thymus Thymoms ++ MC +/-
- 58 66 F Brain Meningioma +/- - - 59 62 M Brain Glioblastoma
multiforme +++ M +++ M - Abbreviations: C: Cytoplasmic staining, *:
Background stain, CS: The section is completely sloughed, PS: The
section is partially sloughed, F: The section is folded, SSC:
Squamous cell carcinoma, HCC: Hepatocellular carcinoma.
Therefore, it appears that the H460-16-2 antigen is not solely
found on the membranes of breast cancers but also on the membrane
of a large variety of tumor types. These results indicate that
H460-16-2 has potential as a therapeutic drug in a wide variety of
tumor types besides breast. Again, the similar yet distinct
staining pattern of H460-16-2 compared to L178 anti-CD44 implies
that H460-16-2 is recognizing an epitope present on a variant of
CD44.
The preponderance of evidence shows that H460-16-2 mediate
anti-cancer effects through ligation of a carbohydrate dependent
conformational epitope present on a variant of CD44. It has been
shown, in example 3, H460-16-2 antibody can be used to
immunoprecipitate the cognate antigen from expressing cells such as
MB-231 cells. Further it could be shown that the H460-16-2 antibody
could be used in detection of cells and/or tissues which express a
CD44 antigenic moiety which specifically binds thereto, utilizing
techniques illustrated by, but not limited to FACS, cell ELISA or
IHC.
Thus, it could be shown that the immunoprecipitated H460-16-2
antigen can inhibit the binding of H460-16-2 to such cells or
tissues using such FACS, cell ELISA or IHC assays. Further, as with
the H460-16-2 antibody, other anti-CD44 antibodies could be used to
immunoprecipitate and isolate other forms of CD44 antigen, and the
antigen can also be used to inhibit the binding of those antibodies
to the cells or tissues that express the antigen using the same
types of assays. It could also be shown that if an anti-CD44
antibody that recognizes all forms of CD44 (i.e. a pan-CD44
antibody) were used to isolate its cognate antigen, then that
antigen could also inhibit the binding of H460-16-2 antigen to
cells or tissues that express that antigen, thus also demonstrating
the binding of H460-16-2 to an epitope of CD44 on cells and tissues
expressing that antigen. Alternatively, a comparison of H460-16-2
and pan-CD44 antibody in assays such as competitive binding assays.
ELISA, cell ELISA, FACS or the like, where both antibodies are
present can also demonstrate the binding of H460-16-2 to an epitope
of CD44 on cells and tissues expressing that antigen.
All patents and publications mentioned in this specification are
indicative of the levels of those skilled in the art to which the
invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
It is to be understood that while a certain form of the invention
is illustrated, it is not to be limited to the specific form or
arrangement of parts herein described and shown. It will be
apparent to those skilled in the art that various changes may be
made without departing from the scope of the invention and the
invention is not to be considered limited to what is shown and
described in the specification. One skilled in the art will readily
appreciate that the present invention is well adapted to carry out
the objects and obtain the ends and advantages mentioned, as well
as those inherent therein. Any oligonucleotides, peptides,
polypeptides, biologically related compounds, methods, procedures
and techniques described herein are presently representative of the
preferred embodiments, are intended to be exemplary and are not
intended as limitations on the scope. Changes therein and other
uses will occur to those skilled in the art which are encompassed
within the spirit of the invention and are defined by the scope of
the appended claims. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in the art are intended to be within the
scope of the following claims.
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